WO2017092663A1 - 小分子组合物诱导人肿瘤细胞直接重编程为非致瘤性细胞的方法 - Google Patents

小分子组合物诱导人肿瘤细胞直接重编程为非致瘤性细胞的方法 Download PDF

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WO2017092663A1
WO2017092663A1 PCT/CN2016/107910 CN2016107910W WO2017092663A1 WO 2017092663 A1 WO2017092663 A1 WO 2017092663A1 CN 2016107910 W CN2016107910 W CN 2016107910W WO 2017092663 A1 WO2017092663 A1 WO 2017092663A1
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tumor
cells
composition
cancer
inhibitor
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PCT/CN2016/107910
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French (fr)
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张培霖
王红阳
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海门雨霖细胞科技有限责任公司
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Priority to US15/780,398 priority Critical patent/US10463641B2/en
Priority to JP2018529098A priority patent/JP2019502671A/ja
Priority to EP16869973.4A priority patent/EP3384929B1/en
Publication of WO2017092663A1 publication Critical patent/WO2017092663A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • A61K31/203—Retinoic acids ; Salts thereof
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/443—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with oxygen as a ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/472—Non-condensed isoquinolines, e.g. papaverine
    • A61K31/4725—Non-condensed isoquinolines, e.g. papaverine containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/30—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from cancer cells, e.g. reversion of tumour cells

Definitions

  • the present invention belongs to the field of oncology, stem cell reprogramming, and pharmaceutical cross-linking; more specifically, the present invention relates to the use of a small molecule composition to induce direct reprogramming (transdifferentiation) of human tumor cells into non-tumorigenic cells with multiple targets.
  • a method for tumor cell apoptosis and a small molecule composition thereof; the small molecule composition may be added with a drug carrier or an excipient to develop a drug or a drug formulation for clinical treatment of a tumor; or an aqueous solvent or an organic solvent, a basal medium or Serum-free medium is prepared as a reagent or medium.
  • Tumors are composed of abnormal cells at various degrees of differentiation and are heterogeneous.
  • Existing radiation therapy (radiotherapy), chemical drug therapy (chemotherapy), targeted therapy, biological immunotherapy, and induction of differentiation and other tumor treatment methods are all aimed at killing tumor cells, and clinical practice proves that it cannot overcome tumor abnormalities.
  • the innovative idea of the invention is to change the killing tumor cells into direct transdifferentiation into normal or non-tumorigenic cells, thereby creating a new treatment method capable of overcoming tumor heterogeneity and high efficiency and low toxicity.
  • Cell reprogramming is the transformation of cells from one type to another by modulating changes in cellular signaling pathways and epigenetics modifications (reprogramming of cells referred to herein to induce cell reprogramming) .
  • Cell reprogramming includes induction of pluripotent cells (iPSCs) reprogramming and direct cell reprogramming (transdifferentiation), which has been widely used for transformation of normal cell types. It has also been reported that individual tumor cells are reprogrammed into iPSCs by exogenous transcription factors (still retaining their tumorigenicity).
  • the present invention does not use any transcription factor, and only uses a chemical small molecule to induce a method for directly regulating reprogramming (transdifferentiation) of tumor cells into non-tumorigenic cells accompanied by apoptosis of tumor cells, and no literature has been reported.
  • the object of the present invention is to provide a multi-target composition for inducing direct reprogramming (transdifferentiation) of human tumor cells into non-tumorigenic cells accompanied by apoptosis of tumor cells (hereinafter referred to as: tumor cell transdifferentiation accompanied by apoptosis) Small molecule composition) and method thereof; the small molecule composition can be developed into a clinical treatment by adding a drug carrier or an excipient a drug or drug formulation of the tumor; or an aqueous or organic solvent, a basal medium or a serum-free medium to prepare a scientific research reagent or medium.
  • the basic mechanism of the present invention is mainly to induce GSK3 ⁇ and TGF ⁇ signaling pathway changes in tumor cells by multi-target combination of GSK3 ⁇ inhibitor and TGF ⁇ inhibitor, and retinoids or compounds which induce epigenetic changes.
  • the synergistic effect regulates the epigenetic changes of tumor cells, reprograms the gene expression profile of tumor cells, thereby transdifferentiating tumor cells into non-tumorigenic cells; while tumor cells that are not transdifferentiated are associated with apoptosis.
  • BMP inhibitors may be added to induce BMP signaling pathway, or BrdU, or EdU may be added, which may have a good effect.
  • GSK3 ⁇ inhibitors and TGF ⁇ inhibitors contain two classes, the same function, or a series of small molecules that induce the same target, and different combinations formed can induce tumor cell transdifferentiation to varying degrees, accompanied by Different degrees of tumor cell apoptosis.
  • BMP inhibitors, retinoids are also similar. Therefore, similar small molecule compounds that have the same function or induce the same target, have the same effect on the same signaling pathway, and a small molecule combination that can induce the direct transdifferentiation of tumor cells into non-tumorigenic cells belong to the present invention. Within the scope of the invention.
  • tumorigenicity is the commonality of tumor cells.
  • the present invention successfully transfects the most heterogeneous hepatoma cells into non-tumorigenic cells, and as a breakthrough point, successively, pancreatic cancer, lung cancer, stomach cancer, breast cancer
  • Various tumors or tumor cells such as lymphoma and glioma, are chemically induced to differentiate into non-tumorigenic cells to varying degrees, with varying degrees of tumor cell apoptosis. Therefore, the "small molecule composition induces direct reprogramming (transdifferentiation) of human tumor cells into non-tumorigenic cells and is accompanied by tumor cell apoptosis and small molecule composition", and the target is liver cancer.
  • a variety of tumors or cells thereof that is, can transform various tumor cells into non-tumorigenic cells to varying degrees, accompanied by different degrees of apoptosis of tumor cells.
  • a small molecule composition for chemically inducing direct reprogramming transformation or transdifferentiation of a human tumor cell into a non-tumorigenic cell accompanied by varying degrees of apoptosis of the tumor cell
  • the composition includes a GSK3 ⁇ inhibitor, a TGF ⁇ inhibitor; or the composition consists of a GSK3 ⁇ inhibitor, a TGF ⁇ inhibitor.
  • the composition comprises:
  • GSK3 ⁇ inhibitor 0.046-4.65 parts by weight
  • TGF ⁇ inhibitor 0.038-7.68 parts by weight.
  • composition can be a solution state composition comprising:
  • GSK3 ⁇ inhibitor final concentration: 0.1-10 uM
  • TGF ⁇ inhibitor final concentration: 0.1-20 uM.
  • composition comprises:
  • GSK3 ⁇ inhibitor 0.232-2.325 parts by weight
  • TGF ⁇ inhibitor 0.192-3.84 parts by weight.
  • composition can be a solution state composition comprising:
  • GSK3 ⁇ inhibitor final concentration: 0.5-5 uM
  • TGF ⁇ inhibitor final concentration: 0.5-10 uM.
  • the GSK3 ⁇ inhibitor, the TGF ⁇ inhibitor is added in an amount of from 0.01 to 99.9% by weight based on the total weight of the composition; for example, from 0.1 to 50% (as in solution) or from 50 to 99.9%. More specifically, such as 1%, 5%, 10%, 20%, 30%, and the like.
  • the weight ratio of the GSK3 ⁇ inhibitor (or GSK3 ⁇ inhibitor CHIR99021), TGF ⁇ inhibitor (or TGF ⁇ inhibitor SB431542 or/and A83-01) in the composition is: (0.046-4.65) ): (0.038-7.68); preferably, (0.232-2.325): (0.192-3.84); or the molar ratio in the solution state is: (0.1-10): (0.1-20); preferably, (0.5) -5): (0.5-10).
  • composition further comprises:
  • Retinoic acid compound 0.03-6.0 parts by weight; preferably 0.15-3 parts by weight.
  • the addition of retinoids can promote and enhance tumor cell transdifferentiation with concomitant apoptosis, or expand the applicable tumor type or range.
  • the composition may be a solution state composition having a final concentration of the retinoid compound of from 0.1 to 20 uM; preferably from 0.5 to 10 uM.
  • the GSK3 ⁇ inhibitor, the TGF ⁇ inhibitor and the retinoid compound are added in an amount of 0.02 to 99.9% by weight based on the total weight of the composition; for example, 0.2 to 50% or 50 to 99.9%. More specifically, such as 1%, 5%, 10%, 20%, 30%, 40%, and the like.
  • the composition is a GSK3 ⁇ inhibitor (or GSK3 ⁇ inhibitor CHIR99021), a TGF ⁇ inhibitor (or TGF ⁇ inhibitor SB431542 or/and A83-01) and a retinoid (or retinoic acid)
  • a GSK3 ⁇ inhibitor or GSK3 ⁇ inhibitor CHIR99021
  • TGF ⁇ inhibitor or TGF ⁇ inhibitor SB431542 or/and A83-01
  • a retinoid or retinoic acid
  • parts by weight (0.046-4.65): (0.038-7.68): (0.03-6.0); preferably, (0.232-2.325): (0.192-3.84): (0.15-3); or
  • composition may also be added to include one or more ingredients selected from the group consisting of:
  • BMP inhibitor (such as LDN-193189): 0.02-4.65 parts by weight; preferably: 0.203-2.03 parts by weight; or
  • BrdU 0.15-30 parts by weight; preferably 1.5-15 parts by weight; or
  • EdU 0.125-25 parts by weight; preferably 1.25-12.5 parts by weight.
  • the composition may be a solution in a solution state, comprising one or more components selected from the group consisting of:
  • the final concentration of the BMP inhibitor is: 0.05-10 uM; preferably 0.5-5 uM;
  • the final concentration of BrdU is: 0.5-100 uM; preferably 5-50 uM; or
  • the final concentration of EdU is: 0.5-100 uM; preferably 5-50 uM.
  • BMP inhibitor such as LDN-193189
  • BrdU or EdU can further promote or enhance the transdifferentiation of some malignant tumor cells with apoptosis.
  • the GSK3 ⁇ inhibitor, the TGF ⁇ inhibitor, the retinoid compound, and/or the BMP inhibitor, and/or BrdU (or/and EdU) are added in an amount of 0.02 to 99.9 by weight based on the total weight of the composition. %; for example, 0.2 ⁇ 50% or 50 to 99.9%. More specifically, such as 1%, 5%, 10%, 20%, 30%, 40%, and the like.
  • the composition is a GSK3 ⁇ inhibitor (such as the GSK3 ⁇ inhibitor CHIR99021), a TGF ⁇ inhibitor (such as the TGF ⁇ inhibitor SB431542 or/and A83-01), a retinoid compound (such as retinoic acid).
  • a GSK3 ⁇ inhibitor such as the GSK3 ⁇ inhibitor CHIR99021
  • a TGF ⁇ inhibitor such as the TGF ⁇ inhibitor SB431542 or/and A83-01
  • a retinoid compound such as retinoic acid
  • BMP inhibitor such as BMP inhibitor LDN-193189
  • BrdU in parts by weight: (0.046-4.65): (0.038-7.68): (0.03-6.0): (0.02-4.65): 0.15-30; Good ground, (0.232-2.325): (0.192-3.84): (0.15-3): (0.203-2.03): (1.5-15) is present; or in the solution state in molar ratio: (0.1-10): (0.1 -20): (0.1-20): (0.05-10): (0.5-100); preferably, (0.5-5): (0.5-10): (0.5-10): (0.5-5): (5-50) exists.
  • the composition is a GSK3 ⁇ inhibitor (such as the GSK3 ⁇ inhibitor CHIR99021), a TGF ⁇ inhibitor (such as the TGF ⁇ inhibitor SB431542 or/and A83-01), a retinoid compound (such as retinoic acid).
  • a GSK3 ⁇ inhibitor such as the GSK3 ⁇ inhibitor CHIR99021
  • a TGF ⁇ inhibitor such as the TGF ⁇ inhibitor SB431542 or/and A83-01
  • a retinoid compound such as retinoic acid
  • BMP inhibitor such as BMP inhibitor LDN-193189
  • EdU in parts by weight: (0.046-4.65): (0.038-7.68): (0.03-6.0): (0.02-4.65): (0.125-25)
  • (0.232-2.325): (0.192-3.84): (0.15-3): (0.203-2.03): (1.25-12.5) is present; or in a solution state in a molar ratio: (0.1-10): (0.1-20): (0.1-20): (0.05-10): (0.5-100); preferably, (0.5-5): (0.5-10): (0.5-10): (0.5-5 ): (5-50) exists.
  • the weight unit of the above weight ratio may be any unit of weight such as kilograms (kg), milligrams (mg), micrograms (ug), etc.; the molar unit of the molar concentration ratio may be: friction (M), millimolar (mM), Micromolar (uM) and other molar concentration units.
  • the effective dosage of the large animal or human (including the solid or solution dosage conversion) is converted into a small animal using the corresponding professional conversion formula.
  • the GSK3 ⁇ inhibitor includes, but is not limited to, CHIR-99021, BIO, IM-12, TWS119, 1-Azakenpaullone, CHIR-98014, Tideglusib, AR-A014418, LY2090314, SB216763, AZD1080, etc.
  • the TGF ⁇ inhibitors include, but are not limited to, SB431542, A83-01, SB525334, LY2109761, RepSox, SD-208, GW788388, SB505124, EW-7197, etc., having the same function, or inducing the same target.
  • the retinoids are natural or synthetic, including but not limited to: retinoic acid (alias: all-trans retinoic acid, ATRA), 13-cis retinoic acid, 9-cis retinoic acid, isotretinoin
  • retinoic acid alias: all-trans retinoic acid, ATRA
  • 13-cis retinoic acid 13-cis retinoic acid
  • 9-cis retinoic acid isotretinoin
  • RA retinoic acid
  • the BMP inhibitors include, but are not limited to, LDO-193189, K02288, DMH1, etc., the same type of BMP signaling pathway inhibitors or compounds that have the same function, or induce the same target, or equivalent agents thereof.
  • the composition is a pharmaceutical composition, further comprising a pharmaceutically acceptable carrier or excipient, the carrier or excipient including, but not limited to, water, saline, phosphate buffer Or other aqueous solvent; DMSO (dimethyl sulfoxide), glycerol and ethanol or other organic solvents; microspheres, liposomes, microemulsions or polymeric surfactants; colloidal drug delivery systems or polymer drug delivery systems; Or preservatives, antioxidants, flavoring agents, fragrances, solubilizers, emulsifiers, pH buffering substances, binders, fillers, lubricants or other pharmaceutical excipients.
  • a pharmaceutically acceptable carrier or excipient including, but not limited to, water, saline, phosphate buffer Or other aqueous solvent; DMSO (dimethyl sulfoxide), glycerol and ethanol or other organic solvents; microspheres, liposomes, microemulsions or polymeric surfactants; coll
  • the pharmaceutical dosage form that can be prepared by the composition includes, but is not limited to, a solid dosage form including, but not limited to, a powder, a powder, a tablet, a pill, a capsule, a sustained release agent, and a control. Immediate release agent; liquid dosage form including, but not limited to, injections, infusion solutions, suspensions, or other liquid dosage forms; gaseous dosage forms; or semi-solid dosage forms.
  • the composition may be formulated into an aqueous reagent or an organic solvent to prepare a scientific research reagent; a basal medium or a serum-free medium may be added to prepare a tumor cell to be directly reprogrammed into a non-tumorigenic cell.
  • a basal medium or a serum-free medium may be added to prepare a tumor cell to be directly reprogrammed into a non-tumorigenic cell.
  • Medium each component in the composition is present in basal cell culture medium containing 5-20% calf serum, 1% streptomycin mixture (100x) or serum-free medium containing various cytokines or growth factors.
  • the cell differentiation basic medium is not included in the composition.
  • compositions for the development or preparation of a medicament (or pharmaceutical formulation) for treating a tumor or for the preparation of a direct reprogramming (transdifferentiation) of a human tumor cell to a non-induced A culture medium or reagent that is associated with tumor cells and is associated with tumor cell apoptosis.
  • a method of inducing direct reprogramming of a tumor cell into a non-tumorigenic cell accompanied by apoptosis of the tumor cell comprising: applying any of claims 1-6
  • the compositions described induce the direct transdifferentiation of human tumor cells into non-tumorigenic cells with concomitant tumor cell apoptosis.
  • a medium or reagent method for preparing a transdifferentiation and apoptosis associated with a human tumor cell, and an experimental procedure thereof comprising:
  • Concentrate reagent preparation The composition according to any one of claims 1 to 6, wherein each component is dissolved in an organic solvent or an aqueous solvent to prepare a concentrate reagent; preferably, the organic solvent comprises two Methyl sulfoxide; preferably, the aqueous solvent comprises: water, physiological saline, phosphate buffer;
  • the concentrate reagent in step (1) is separately diluted into a basic cell culture medium containing 5-20% calf serum, 1% streptomycin mixed solution (100x) or contains various cells.
  • a serum-free medium of a factor or a growth factor (such that the concentration of each component conforms to the concentration defined in the composition according to any one of claims 1 to 6), and a medium for inducing tumor cell transdifferentiation with apoptosis is obtained;
  • the percentage of each component of the medium can also be up and down by 50%; preferably up and down by 30%; more preferably up and down by 20%, such as 10%, 5%;
  • kits/kit for inducing direct reprogramming of a human tumor cell into a non-tumorigenic cell accompanied by apoptosis of the tumor cell, the kit comprising: A composition according to any of the preceding claims; or a medicament or pharmaceutical formulation for the treatment of a tumor prepared based on the composition; or a scientific research reagent or medium prepared based on the composition.
  • the tumor or tumor cell includes, but is not limited to, liver cancer, lung cancer, gastric cancer, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, osteosarcoma, lymphoma, leukemia, nasopharyngeal Cancer, esophageal cancer, cervical cancer, oral cancer, salivary gland tumor, nasal and paranasal sinus malignancy, laryngeal cancer, ear tumor, ocular tumor, thyroid tumor, mediastinal tumor, chest wall, pleural tumor, small intestine tumor, biliary tract tumor, Pancreas and periampullary tumor, mesenteric and retroperitoneal tumor, kidney tumor, adrenal tumor, bladder tumor, testicular tumor, penile cancer, endometrial cancer, ovarian malignant tumor, malignant trophoblastic tumor, vulvar cancer and vaginal cancer, malignant lymph Tumor, multiple myeloma, soft tissue tumor, bone tumor, skin and accessory tumor
  • malignant lymph Tumor
  • FIG. 1 Small molecule (medium 6) induced transdifferentiation of liver cancer cell SMMC-7721 into non-tumorigenic cells with apoptosis.
  • liver cancer cell SMMC-7721 was induced to transdifferentiate into non-tumorigenic hepatocyte-like cells by medium 6 for 10 days, and the morphology was completely changed, indicating that it had been transdifferentiated;
  • liver cancer cell SMMC-7721 was induced to undergo transdifferentiation by medium 6 with apoptosis statistics for 1-14 days of apoptosis culture.
  • the blue column represents early apoptosis
  • the red column represents late (Late) apoptosis
  • T, T1W, T2W represent the apoptosis of SMMC-7721 cells treated directly and treated for 1 and 2 weeks, respectively.
  • Control group and treatment group Apoptotic values were statistically significant (p ⁇ 0.05).
  • Hepatoma cell HepG2 was induced to transdifferentiate by medium 4, 1 with apoptosis.
  • Hepatoma cell HepG2 was induced to transdifferentiate into non-tumorigenic hepatocyte-like cells by medium for 8 days, and the morphology was completely changed, indicating that it had been transdifferentiated;
  • Hepatoma cell HepG2 was induced to undergo transdifferentiation with apoptosis by medium 1.
  • the blue column represents early apoptosis
  • the red column represents late apoptosis
  • T, T1W, and T3W represent the results of apoptosis detection directly treated and treated for 1 and 3 weeks, respectively.
  • the apoptosis values of the control group and the treatment group were statistically different (p ⁇ 0.05).
  • Fig. 3 5-Fu-resistant liver cancer cells 7402/5-Fu were induced to undergo apoptosis by medium 5 and 2.
  • liver cancer cells 7402/5-Fu were treated with medium 5 for 2 weeks to induce complete changes in morphology after transdifferentiation, indicating that it has been transdifferentiated;
  • liver cancer cells 7402/5-Fu were induced to undergo transdifferentiation with apoptosis in culture medium 2.
  • the blue column represents early apoptosis
  • the red column represents late apoptosis
  • T2W, T3W, and T4W represent the results of apoptosis detection at 2, 3, and 4 weeks, respectively.
  • the apoptosis values of the control group and the treatment group were statistically different (p ⁇ 0.005).
  • FIG. 1 Liver cell SMMC-7721 (medium 6), HepG2 (medium 4), 7402/5-Fu (medium 5) were induced to transdifferentiate into non-tumorigenic hepatocyte-like cells with normal hepatocyte function.
  • PAS glycogen staining
  • Oil-red oil red staining, reflecting the fat intake function.
  • Liver cell SMMC-7721 (medium 10), HepG2 (medium 11), 7402/5-Fu (medium 12) were induced to transdifferentiate into non-tumorigenic hepatocyte-like cells with normal hepatocyte white Protein secretion (ALB), urea production (Urea), CYP1A2 induction and CYP3A4 induction.
  • ALB normal hepatocyte white Protein secretion
  • Urea urea production
  • CYP1A2 induction CYP3A4 induction.
  • the non-tumorigenic hepatocyte-like cells obtained by transdifferentiating the hepatoma cell SMMC-7721 by medium 10 have normal hepatocyte function;
  • the non-tumorigenic hepatocyte-like cells obtained by transdifferentiation of hepatoma cell HepG2 by medium 11 have normal hepatocyte function;
  • Non-tumorigenic hepatocyte-like cells obtained by transducing differentiation of 5-Fu liver cancer cells 7402/5-Fu by medium 12 have normal hepatocyte function;
  • T1W, T2W, and T3W represent treatments of 1, 2, and 3 weeks, respectively.
  • Rif Rifampin
  • Ome Ogilto.
  • Non-tumorigenic hepatocyte-like cells obtained by transdifferentiation of hepatoma cells SMMC-7721 (medium 6), HepG2 (medium 4) and 7402/5-Fu (medium 5) are no longer in vitro or in vivo. It is tumorigenic.
  • liver cancer cells SMMC-7721, HepG2 and 7402/5-Fu were not cloned by non-tumorigenic hepatocyte-like cells induced by transdifferentiation in culture medium 6, 4, 5, respectively, and lost tumorigenicity in vitro;
  • Non-tumorigenic hepatocyte-like cells obtained by transdifferentiation of hepatoma cell SMMC-7721 did not produce tumors in vivo (treatment for 4 weeks), and tumorigenicity was lost in vivo (medium 6).
  • liver cancer 7402/5-Fu induced non-tumorigenic hepatocyte-like cells obtained by transdifferentiation did not produce tumors in vivo (treatment for 4 weeks), loss of tumorigenicity in vivo (medium 5);
  • the cells in the treatment group did not form a tumor, and the tumorigenicity was lost;
  • the cells in the left control group of the hind leg formed a tumor;
  • C The figure below shows the anatomical shape of the tumor formed by the control group. Exterior.
  • FIG. 7 Small molecule combination applied to patient liver cancer tissue PDX animal model test (transdifferentiation with apoptotic composition 8). The results showed that the tumor tissue of the treatment group was necrotic, the tissue structure was destroyed or lost (red staining); the tumor tissue and cell structure of the control group did not change (purple staining) (PDX-80872).
  • Figures 4, 7, 8 and the like represent mouse ear stud codes.
  • Fig. 8 Normal human fibroblasts and hepatocytes were cultured for 3 weeks (transdifferentiation with apoptosis medium 8, 3), and the morphology was unchanged, indicating that they were not affected.
  • FIG. 9 Nasopharyngeal carcinoma cell line HNE and lung cancer cell H460 were induced to undergo transdifferentiation with apoptosis by medium 9,7, respectively. It can be seen from Fig. 9 that the morphology of the cancer cells treated with HNE (medium 9 treatment) in the nasopharyngeal carcinoma cells completely changed after transdifferentiation, indicating that they have been transdifferentiated; the treated group lung cancer cells H460 were treated separately (media 9, 7), among which Lung cancer cell H460 was induced almost exclusively by (medium 7) (green column represents early apoptosis, red column represents late apoptosis; T10D, T20D represents treatment for 10 days, treatment for 20 days); and control group lung cancer cells H460 has almost no apoptosis.
  • Gastric cancer cells SGC-7901 and MKN28 were induced to transdifferentiate (media 12, 13 treatment for 2 weeks).
  • Gastric cancer cells SGC-7901 (medium 12) and MKN28 (medium 13) were treated for 2 weeks, respectively, and the morphology of the cancer cells in the treated group was completely transformed after transdifferentiation, indicating that it had been transdifferentiated;
  • Pancreatic cancer cells SW1990 were induced to transdifferentiate (medium 12 treatment for 2 weeks). After transdifferentiation of cancer cells in the pancreatic cancer cell SW1990 treated group, the morphology completely changed, indicating that it has been transdifferentiated;
  • FIG. 12 Breast cancer cell SKBr3 was induced to transdifferentiate (medium 13 treatment for 2 weeks). After transdifferentiation of cancer cells in the SKBr3-treated group of breast cancer cells, the morphology was completely changed, indicating that it had been transdifferentiated;
  • Leukemia cell U937, B cell lymphoma SUDHL-4 was induced to undergo transdifferentiation with apoptosis (medium 10, 11 treatment for 10 days). Most of the cancer cells in the leukemia cells U937 (medium 10) and B-cell lymphoma SUDHL-4 (medium 11) were induced to undergo apoptosis, and the cell morphology was no longer intact.
  • FIG. 14 Breast cancer cell SKBr3 and gastric cancer cell MKN28 were induced to transdifferentiate (medium 13, 14). Breast cancer cells SKBr3 and gastric cancer cells MKN28 were treated with medium 13, 14 for 2 weeks, respectively.
  • the upper right panel of Fig. 14 shows that the morphology of cancer cells in the treatment group (T) was completely changed, indicating that it had been transdifferentiated.
  • FIG. 15 Small molecule combination induces tumorigenicity after transdifferentiation of intestinal cancer HCT116 cells.
  • Intestinal cancer cell HCT116 was treated with medium 9 for 2 weeks.
  • the right panel showed that the morphology of cancer cells in the HCT116-treated group was completely changed, and clones were no longer formed, indicating that they had transdifferentiated and lost tumorigenicity.
  • Figure 16 Small molecule induced loss of tumorigenicity after transdifferentiation of prostate cancer PC-3 cells.
  • Prostate cancer PC-3 cells were treated with medium 5 for 2 weeks, and the right panel of Fig. 16 showed that the pancreatic cancer PC-3 cells in the treated group were no longer formed into clones after induction transdifferentiation, and the tumorigenicity in vitro was lost.
  • FIG. 17 Small molecule induced loss of tumorigenicity after transdifferentiation of ovarian cancer cells SKOV3 and A2780 cells.
  • the upper and lower graphs respectively show that the ovarian cancer cells SKOV3 and A2780 in the treatment group were no longer formed into clones after being induced to differentiate by the medium 7, 8 and lost tumorigenicity.
  • FIG. 18 Small molecule induces tumorigenicity after transdifferentiation of gastric cancer and breast cancer cells.
  • the top and bottom images are displayed separately Gastric cancer cells MKN28 and breast cancer cells SKbr3 in the treatment group were treated with medium 10 and 11 for 2 weeks, respectively, and no clones were formed after transdifferentiation, and tumorigenicity was lost.
  • FIG. 19 Small molecules induce complete changes in morphology after glioma cell transdifferentiation.
  • the glioma cells T98G and U87MG in the right upper and lower images were treated with medium 4 and 5 for 2 weeks, respectively, and the morphology was completely changed after transdifferentiation, indicating that they had been transdifferentiated.
  • FIG. 20 Small molecule combination applied to liver cancer PDX animal model experiment.
  • the liver cancer tissues and cells in the left-hand treatment group were treated with the injection reagent 21 prepared by transdifferentiation with the apoptotic small molecule composition 21 for 3 weeks, and a large area was necrotic, and the cancer tissue structure was destroyed or lost.
  • the treatment group on the right shows that after treatment with small molecule reagent for 3 weeks, residual tissue and cells express HNF4a, a unique marker of human hepatocytes, suggesting that transdifferentiation has occurred.
  • FIG. 21 Small molecule induces tumorigenicity in vitro and in vivo after transdifferentiation of lung cancer cells.
  • A was treated with medium for 2 weeks. The following group showed that lung cancer cells A549, H1299 and H460 were no longer formed into clones after induction by medium 14, 13, and 9 respectively, and lost tumorigenicity in vitro;
  • B naked The right hind leg of the mouse (indicated by the blue arrow) was injected into the nude mice for 4 weeks after transdifferentiation of the treated group.
  • the treated group showed that the cells after transdifferentiation no longer formed tumors, indicating that the lung cancer cells A549 (medium 14 treatment) After being induced to transdifferentiate, the body also loses tumorigenicity.
  • the red arrow refers to a tumor formed by untreated lung cancer cells.
  • FIG 22 Small cell induced lung cancer cell H1299 transdifferentiation, lost tumorigenicity in vitro.
  • the right panel shows that the lung cancer cells H1299 in the treated group (medium 15 treated for 2 weeks) no longer formed clones after transdifferentiation, and lost tumorigenicity.
  • FIG. 23 Ovarian cancer cells A2780 and SKOV3 were induced to lose tumorigenicity in vitro after transdifferentiation.
  • the upper and lower images respectively showed that the treated group of ovarian cancer cells A2780 (medium 16) and SKOV3 (medium 17) were treated for 2 weeks, and no clones were formed after transdifferentiation, and tumorigenicity was lost.
  • FIG. 24 Prostate cancer cell PC9 was treated with medium 18 for 2 weeks to induce transdifferentiation. After the prostate cancer cell line PC9 in the right-handling group was induced to transdifferentiate, the cell morphology completely changed, indicating that it had been transdifferentiated.
  • FIG. 25 Gastric cancer SGC-7901 cells were induced to transdifferentiate by mediums 19 and 20, respectively.
  • the A-treatment group showed that gastric cancer SGC-7901 cells (treated for 2 weeks in medium 19) no longer formed clones after transdifferentiation, and lost tumorigenicity in vitro.
  • B shows the statistical results of gastric cancer SGC-7901 cells induced apoptosis (medium 20 treatment for 2 weeks).
  • the green column represents early apoptosis (Early)
  • the red column represents late apoptosis (Late)
  • T1W and T2W represent the apoptosis statistics of 1 and 2 weeks, respectively; and the control group has few early and late natural apoptosis.
  • the apoptosis values of the control group and the treatment group were statistically different (p ⁇ 0.005).
  • pancreatic cancer SW1990 cells were treated with medium 16 for 2 weeks to induce tumorigenicity after transdifferentiation.
  • the pancreatic cancer SW1990 cells in the right-treated group no longer formed clones after transdifferentiation and lost tumorigenicity in vitro.
  • the inventors have conducted intensive research to reveal a method for small cell composition to induce direct reprogramming (transdifferentiation) of human tumor cells into non-tumorigenic cells accompanied by apoptosis of tumor cells and small molecule compositions thereof;
  • the molecular composition may be added with a drug carrier or an excipient to develop a drug or a pharmaceutical formulation for clinical treatment of a tumor;
  • Solvent or organic solvent, basal medium or serum-free medium is prepared as a scientific research reagent or medium.
  • the small molecule composition induces direct reprogramming (transdifferentiation) of human tumor cells.
  • the method is first applied to liver cancer cells, and the liver cancer cells are directly transdifferentiated into non-tumorigenic hepatocyte-like cells accompanied by different degrees of apoptosis of liver cancer cells;
  • the obtained hepatocyte-like cells have normal hepatocyte function; there is no damage to normal hepatocytes and fibroblasts within the experimental test range.
  • the small molecule composition also transfects various tumor or tumor cells represented by lung cancer, gastric cancer, pancreatic cancer, breast cancer, leukemia, lymphoma, glioma, and the like into non-tumorigenic cells. And accompanied by varying degrees of tumor cell apoptosis.
  • Chemically induced cell direct reprogramming refers to the process of altering cell fate by regulating cellular signaling pathways and epigenetic changes without altering the gene sequence.
  • iPSC chemically induced pluripotent cells
  • the reprogramming initiation and target cell types are also reprogrammed from differentiated cells to pluripotent stem cells, extended to direct reprogramming to another differentiated cell; even transcription factors can be introduced exogenously from abnormal cells (myxoma cells) Programming for versatility Like cells, but still retain their tumorigenicity (Zhang X, Cruz FD, Terry M, Remotti F and Matushansky I. Terminal differentiation and loss of tumorigenicity of human cancers via pluripotency-based reprogramming. Oncogene, 2, 2249–2260, 2013) Therefore, it has become possible to directly reprogram tumor cells into non-tumorigenic cells using only small molecules.
  • the invention first selects the liver cancer with the strongest heterogeneity as a breakthrough point. Screening of small molecule combinations capable of constructing and maintaining normal (non-tumorigenic) hepatocytes (target cells) and capable of breaking through various "energy barriers" in the process of transdifferentiation of liver cancer cells to normal liver cells
  • a small molecule induces transdifferentiation of hepatoma cells into non-tumorigenic hepatocytes, accompanied by different degrees of apoptosis of hepatocellular carcinoma cells;
  • this innovative method has the following advantages: 1 small molecule properties are stable, time, dose and combination are easy to apply Control, the effect is stable and reliable; 2 Hepatocyte-like cells after transdifferentiation not only have normal mature human hepatocyte function, but also no tumorigenicity in vivo and in vitro; 3 This method can strongly transdifferentiate liver cancer cells and is normal Hepatocytes and fibroblasts are not damaged; no cell killing reagent is needed to kill tumor cells, thus avoiding the
  • the method is of great significance for the control or effective treatment of human liver cancer with complicated pathogenicity and pathogenesis, heterogeneity and lack of effective treatment.
  • the small molecule composition also induces nasopharyngeal carcinoma, lung cancer, gastric cancer, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, osteosarcoma, lymphoma, leukemia and other blood.
  • Systemic tumors and substantial tumor cells are directly reprogrammed (transdifferentiated) into non-tumorigenic cells with concomitant apoptosis of tumor cells with the same or similar effects.
  • the inventors have extensively studied for the first time to simultaneously inhibit the GSK3 ⁇ and TGF ⁇ signaling pathways to induce tumor cell transdifferentiation and accompany tumor cell apoptosis. According to the individual differences in tumor cell heterogeneity or the difference between tumor cell lines, the ratio of the number of tumor cells induced to transdifferentiation to the number of tumor cells induced to induce apoptosis varies somewhat; some are biased towards transdifferentiation, and some are biased towards Apoptosis; while inhibiting GSK3 ⁇ and TGF ⁇ signaling pathways favored apoptosis, transdifferentiation was not thorough enough.
  • Inhibition of GSK3 ⁇ and TGF ⁇ signaling pathways combined with the coordination of retinoic acid (RA) compounds can enhance the differentiation of tumor cells into non-tumorigenic cells induced by small molecule compositions, accompanied by different degrees of apoptosis in tumor cells. . If combined with the down-regulation of the BMP signal path, and/or in conjunction with BrdU (or / and EdU), then Some of the more malignant tumor cells can be further enhanced or promoted to accompany tumor cell apoptosis. Although the proportion of transdifferentiation and apoptosis by small molecule combinations is different due to the heterogeneity of tumors.
  • the small molecule composition has the potential to be developed as a new drug or drug formulation for anti-tumor therapy; and can be directly prepared as a research medium for chemically inducing tumor cells to be directly reprogrammed into non-tumorigenic cells accompanied by tumor cell apoptosis. Or reagent.
  • RA retinoic acid
  • the term "consisting essentially of” means that in the composition, in addition to containing the essential ingredients or essential components, minor amounts and/or impurities which do not affect the active ingredient may be contained.
  • sweeteners may be included to improve taste, antioxidants to prevent oxidation, and other pharmaceutical additives, carriers, excipients commonly used in the art.
  • the term "pharmaceutically acceptable” ingredient is a substance that is suitable for use in humans and/or animals without excessive adverse side effects (eg, toxicity, irritation, and allergies), ie, having a reasonable benefit/risk ratio;
  • a pharmaceutical carrier or excipient as is conventional in the art.
  • the term "effective amount” refers to an amount that is functional or active to a human and/or animal and that is acceptable to humans and/or animals.
  • pharmaceutically acceptable carrier or excipient refers to a system that alters the manner in which the drug enters the body and its distribution in the body, controls the rate of drug release, and delivers the drug to the targeted organ;
  • the pharmaceutical carrier itself is not an essential active ingredient and is not excessively toxic after administration.
  • Suitable carriers are well known to those of ordinary skill in the art and include, but are not limited to, water, saline, phosphate buffers, and other aqueous solvents; DMSO (dimethyl sulfoxide), glycerol and ethanol, and other organic solvents; microspheres, Liposomes, microemulsions, polymeric surfactants; colloidal drug-loading systems, novel polymer drug-loading systems, novel drug carriers, and other pharmaceutically acceptable carriers; wherein the excipients are in addition to the main drug in the pharmaceutical preparations Add-ons, also known as excipients.
  • excipients The general requirement for excipients is that it is stable in nature, has no compatibility contraindications with the main drug, does not cause side effects, does not affect the curative effect, and is not easily deformed, chrysolate, mildewed, insects, harmless to the human body, and has no physiological effect at normal temperature. Produce chemistry with the main drug Or physical action, does not affect the determination of the content of the main drug.
  • the carrier or excipient includes, but is not limited to, water, saline, phosphate buffer, etc.; DMSO (dimethyl sulfoxide), organic solvents such as glycerin and ethanol; microspheres, liposomes, microemulsions, polymer surfaces Active agent; colloidal drug-loading system, novel polymer drug-loading system, novel drug carrier and other pharmaceutically acceptable carriers; preservatives, antioxidants, flavoring agents, fragrances, solubilizers, emulsifiers in liquid preparations, pH buffer substances, binders, fillers, lubricants and other pharmaceutical excipients in tablets.
  • DMSO dimethyl sulfoxide
  • organic solvents such as glycerin and ethanol
  • microspheres, liposomes, microemulsions, polymer surfaces Active agent colloidal drug-loading system, novel polymer drug-loading system, novel drug carrier and other pharmaceutically acceptable carriers
  • the pharmaceutical dosage form in the term "pharmaceutical dosage form which can be prepared by the composition” refers to a pharmaceutical application form prepared to meet the needs of treatment or prevention, referred to as a pharmaceutical dosage form; a pharmaceutical dosage form which can be prepared by any of the compositions of the present invention. Including but not limited to: powders, powders, tablets, pills, capsules, sustained release agents, controlled release agents and other solid dosage forms; injections, infusion solutions, suspensions and other liquid dosage forms, as well as gaseous dosage forms, semi-solid dosage forms Other dosage forms.
  • parts by weight or “parts by weight” are used interchangeably and may be any fixed amount expressed in micrograms, milligrams, grams or kilograms (eg, 1 ug, 1 mg, 1 g, 2g, 5g, or kg, etc.).
  • a composition consisting of 1 part by weight of component a and 9 parts by weight of component b may be 1 gram of component a + 9 gram of component b, or may be 10 gram of component a + 90 gram of component b.
  • the above “parts by weight” may be converted into “moles”; and the “parts by weight” may be converted into “molar ratio”.
  • the weight unit of the weight ratio may be: kilogram (kg), milligram (mg), microgram (ug) and the like, and the molar unit of the molar ratio may be: (M), millimolar ( mM), micromolar (uM) and other molar concentration units;
  • the composition is a GSK3 ⁇ inhibitor (or GSK3 ⁇ inhibitor CHIR99021), a TGF ⁇ inhibitor (or TGF ⁇ inhibitor SB431542 or/and A83-01) in parts by weight: (0.046-4.65) : (0.038-7.68); preferably, (0.232-2.325): (0.192-3.84) is present; or in a molar ratio: (0.1-10): (0.1-20); preferably, (0.5-5) :(0.5-10) exists.
  • the composition a GSK3 ⁇ inhibitor (or GSK3 ⁇ inhibitor CHIR99021), a TGF ⁇ inhibitor (or TGF ⁇ inhibitor SB431542 or/and A83-01), and a retinoid compound (or retinoic acid)
  • a GSK3 ⁇ inhibitor or GSK3 ⁇ inhibitor CHIR99021
  • a TGF ⁇ inhibitor or TGF ⁇ inhibitor SB431542 or/and A83-01
  • a retinoid compound or retinoic acid
  • parts by weight (0.046-4.65): (0.038-7.68): (0.03-6.0); preferably, (0.232-2.325): (0.192-3.84): (0.15-3) is present; or in moles Ratio: (0.1-10): (0.1-20): (0.1-20); preferably, (0.5-5): (0.5-10): (0.5-10) is present.
  • the composition is a GSK3 ⁇ inhibitor (such as the GSK3 ⁇ inhibitor CHIR99021), a TGF ⁇ inhibitor (such as the TGF ⁇ inhibitor SB431542 or/and A83-01), a retinoid compound (such as retinoic acid).
  • a GSK3 ⁇ inhibitor such as the GSK3 ⁇ inhibitor CHIR99021
  • a TGF ⁇ inhibitor such as the TGF ⁇ inhibitor SB431542 or/and A83-01
  • a retinoid compound such as retinoic acid
  • BMP inhibitor such as BMP inhibitor LDN-193189
  • BrdU in parts by weight: (0.046-4.65): (0.038-7.68): (0.03-6.0): (0.02-4.65): (0.15-30); preferably, (0.232-2.325): (0.192-3.84): (0.15-3): (0.203-2.03) ): (1.5-15) is present; or in a solution state in a molar ratio: (0.1-10): (0.1-20): (0.1-20): (0.05-10): (0.5-100); preferably , (0.5-5): (0.5-10): (0.5-10): (0.5-5): (5-50) is present.
  • the composition is a GSK3 ⁇ inhibitor (such as the GSK3 ⁇ inhibitor CHIR99021), a TGF ⁇ inhibitor (such as the TGF ⁇ inhibitor SB431542 or/and A83-01), a retinoid compound (such as retinoic acid).
  • a GSK3 ⁇ inhibitor such as the GSK3 ⁇ inhibitor CHIR99021
  • a TGF ⁇ inhibitor such as the TGF ⁇ inhibitor SB431542 or/and A83-01
  • a retinoid compound such as retinoic acid
  • BMP inhibitor such as BMP inhibitor LDN-193189
  • EdU in parts by weight: (0.046-4.65): (0.038-7.68): (0.03-6.0): (0.02-4.65): (0.125-25)
  • (0.232-2.325): (0.192-3.84): (0.15-3): (0.203-2.03): (1.25-12.5) is present; or in a solution state in a molar ratio: (0.1-10): (0.1-20): (0.1-20): (0.05-10): (0.5-100); preferably, (0.5-5): (0.5-10): (0.5-10): (0.5-5 ): (5-50) exists.
  • the components included in the composition and the parts by weight or molar concentration thereof are shown in Table 2 or Table 3 (solution state composition).
  • Tables 1 and 2 can be used as a reference guide. It will be understood, however, that when used in the development of pharmaceutical compositions, the effective dosage of the composition employed will depend on the mode of administration and the type of tumor to be treated and the disease. Change in severity. Moreover, when used in vivo, "weight/kg (body weight)" is usually used as a dosage unit; when the small molecule composition is applied to a large animal and a tumor patient, it is converted according to the dose of the small animal by a corresponding professional conversion formula. The effective use of large animals or humans (including solid or solution dosage conversion) is also within the scope of the present invention.
  • GSK3 ⁇ inhibitor refers to a general term for inhibitors capable of inhibiting the GSK3 ⁇ signaling pathway in cells, including but not limited to: CHIR-99021, BIO, IM-12, TWS119, etc.
  • CHIR-99021 (CT99021), which is a GSK-3 ⁇ and ⁇ inhibitor with IC50 of 10 nM and 6.7 nM, respectively, which is 500 times more potent than CDC2, ERK2 and other kinases;
  • CHIR-99021 (CT99021) HCl a hydrochloride salt of CHIR-99021, is a GSK-3 ⁇ / ⁇ inhibitor with an IC50 of 10 nM/6.7 nM in a cell-free assay, which can be used to distinguish between GSK-3 and its most Close homologs Cdc2 and ERK2;
  • BIO which is a specific GSK-3 inhibitor
  • the IC50 of GSK-3 ⁇ / ⁇ in a cell-free assay is 5 nM
  • IM-12 a selective GSK-3 ⁇ inhibitor with an IC50 of 53 nM, enhances the Wnt signaling pathway
  • TWS119 which is a GSK-3 ⁇ inhibitor with an IC50 of 30 nM in a cell-free assay
  • 1-Azakenpaullone a highly selective GSK-3 ⁇ inhibitor with an IC50 of 18 nM
  • CHIR-98014 which is a potent GSK-3 ⁇ / ⁇ inhibitor with an IC50 of 0.65 nM/0.58 nM in a cell-free assay
  • AR-A014418 an ATP competitive and selective GSK3 ⁇ inhibitor with IC50 and Ki of 104 nM and 38 nM in a cell-free assay
  • LY2090314 which is a potent GSK-3 inhibitor, acting on GSK-3 ⁇ / ⁇ with an IC50 of 1.5 nM/0.9 nM;
  • SB216763 which is a potent, selective GSK-3 ⁇ / ⁇ inhibitor with an IC50 of 34.3 nM;
  • AZD1080 an orally bioavailable, selective, brain-permeable GSK3 inhibitor that inhibits human GSK3 ⁇ and GSK3 ⁇ , Ki of 6.9 nM and 31 nM, respectively, is more selective than CDK2, CDK5, CDK1 and Erk2 14 times higher.
  • the GSK3 ⁇ inhibitor is CHIR-99021, which is nicknamed CT99021; and its molecular structural formula is as shown in the following formula (I):
  • TGF ⁇ inhibitor refers to a general term for inhibitors capable of inhibiting the TGF ⁇ signaling pathway in cells, including but not limited to: SB431542, A83-01, SB525334, LY2109761, RepSox, etc.
  • SB431542, A83-01, SB525334, LY2109761, RepSox, etc. The same function, or the same class of TGF ⁇ signaling pathway inhibitors that induce the same target:
  • SB-431542 a potent, selective ALK5 inhibitor with an IC50 of 94 nM, which is 100 times more potent than p38, MAPK and other kinases;
  • A83-01 which is an inhibitor of ALK5, ALK4 and ALK7 with IC50 of 12, 45 and 7.5 nM, respectively;
  • SB525334 a potent, selective TGF ⁇ receptor I (ALK5) inhibitor with an IC50 of 14.3 nM in a cell-free assay, 4 times lower than ALK5 and no activity against ALK2, 3, and 6;
  • LY2109761 which is a novel, selective TGF- ⁇ receptor type I/II (T ⁇ RI/II) dual inhibitor, with a Ki of 38 nM and 300 nM in a cell-free assay, respectively;
  • RepSox a potent, selective TGF ⁇ R-1/ALK5 inhibitor, binds ATP to ALK5 and ALK5 autophosphorylate, with IC50 of 23 nM and 4 nM in cell-free assays, respectively.
  • SD-208 a selective TGF- ⁇ RI (ALK5) inhibitor with an IC50 of 48 nM and a selectivity more than 100-fold higher than TGF- ⁇ RII;
  • GW788388 a potent, selective ALK5 inhibitor with an IC50 of 18 nM in a cell-free assay, also inhibits TGF-beta type II receptor and activin type II receptor activity, but does not inhibit BMP type II receptor;
  • SB505124 a selective TGF ⁇ R inhibitor that acts on ALK4 and ALK5, with IC50 of 129 nM and 47 nM in cell-free assays, also inhibits ALK7, but does not inhibit ALK1, 2, 3 or 6;
  • EW-7197 a highly potent, selective, orally bioavailable TGF- ⁇ receptor ALK4/ALK5 inhibitor with IC50 of 13 nM and 11 nM, respectively.
  • the TGF ⁇ inhibitor is SB 431542 (or SB-431542); and its molecular structural formula is as shown in the following formula (II):
  • the TGF ⁇ inhibitor is A83-01 (or referred to as A8301); and its molecular structural formula is as shown in the following formula (III):
  • the retinoids compounds include, but are not limited to, Retinoic acid (RA), alias: all trans retinoic acid (ATRA); 13-cis Retinoic acid (13-cis retinoic acid, 13-CRA), 9-cis-retinoic acid (9-CRA), etc.
  • RA Retinoic acid
  • ATRA all trans retinoic acid
  • 13-cis Retinoic acid 13-cis retinoic acid
  • 9-CRA 9-cis-retinoic acid
  • Retinoids have functions that regulate cell proliferation, differentiation, and apo ptosi s, as they activate the corresponding retinoic acid receptor (RAR) and retinoic acid X-nucleus Retinoid x receptor (RXR) protein, by specifically binding to retinoic acid respo nse elements (RARE), regulates the transcriptional activity of specific nuclear genes and produces biological effects.
  • RAR retinoic acid receptor
  • RXR retinoic acid X-nucleus Retinoid x receptor
  • RARE retinoic acid respo nse elements
  • Many of the retinoids and their isomer derivatives have the same or similar functions and thus become an important class of inducing differentiation agents or compounds.
  • Retinoids are a group of oxidative metabolites or derivatives of vitamin A (retinol) and synthetic structures with similar structures to vitamin A, both natural and synthetic.
  • Mainly include: Retinoic acid (RA) (alias: retinoic acid, all-trans retinoic acid), 13-cis retinoic acid (13-CRA), acitretin, 9- 9-cis-retinoic acid (9-CRA), UAB7, UAB8, isotretinoin, fenretinide, acitretin, etretinate , Tazarotene, Adalalene, TTNPB, 3-methyl-TTN PB, AM80, AM580, CD437, Targretin, LGD1069 and other retinoids with the same function.
  • RA Retinoic acid
  • RA Retinoic acid
  • retinoic acid alias: retinoic acid, all-trans retinoic acid
  • 13-CRA 13-cis
  • RA or ATRA TTNPB, AM80 and AM580 are specific agonists of RAR; LGD 1069 and SR 11237 are specific agonists of RXR; 9-CRA and 3-methyl-TTNPB can agonize these two receptor proteins , belongs to the general agonist.
  • the retinoic acid (RA), alias: all trans retinoic acid (ATRA), retinoic acid, vitamin A acid, vitamin formic acid, retinoic acid, All-trans retinoic acid and retinoic acid have a molecular structural formula as shown in the following formula (IV):
  • BMP inhibitor refers to a general term for inhibitors capable of inhibiting BMP signaling pathways in cells, including but not limited to: LDN-193189, LDN193189HCl, K02288, DMH1, etc., having the same function. , or the same class of BMP signaling pathway inhibitors that induce the same target:
  • LDN-193189 a selective inhibitor of BMP signaling pathway, inhibits the transcriptional activities of BMP type I receptors ALK2 and ALK3, with IC50 of 5 nM and 30 nM in C2C12 cells, respectively, acting on BMP specificity in TGF- ⁇ selection
  • the sex is 200 times higher.
  • LDN193189HCl a hydrochloride salt of LDN193189
  • IC50 5 nM and 30 nM in C2C12 cells, respectively, and selectivity for BMP It is 200 times more selective for TGF- ⁇ .
  • DMH1 a selective BMP receptor inhibitor that inhibits ALK2 with an IC50 of 107.9 nM, for AMPK, ALK5, KDR (VEGFR-2) and PDGFR have no inhibitory effect.
  • the BMP inhibitor is LDN-193189 (or LD-N193189); and its molecular structural formula is as shown in the following formula (V):
  • the BrdU Chinese full name 5-bromodeoxyuridine, alias: 5-bromo-2'-deoxyuridine; 5-bromo-2-deoxyuridine; 5-bromo-1 -(2-deoxy- ⁇ -D-ribofuranose) uracil; 5-bromodeoxyuridine; brominated deoxyuridine; English name: 5-Bromo-2"-Deoxyuridine; abbreviation: BrdU or 5-BrdU.
  • the EdU Chinese name: 5-ethynyl-2'-deoxyuridine; alias: 5-ethynyl-2-deoxyuridine; 5-ethynyl-2'-deoxyuridine ; ethynyl-deoxyuridine; English name: 5-Ethynyl-2'-deoxyuridine, abbreviated as EdU; alias: EYdU; Uridine; 5-Ethynyl-durd; 5-Ethynyl-2'-dU;2'deoxyuridine;-ethynyl;2'-deoxy-5-ethynyluridine;2'-deoxy-5-ethynyl-uridin; EdU is a novel thymidine analog that can be incorporated into dividing cells.
  • EdU can be detected in a fluorescent azide, which forms a covalent bond with the former by clicking chemical. Unlike the commonly used bromodeoxyuridine, EdU can be detected without the need for heat or acid treatment. It is commonly used as a cell marker, and recent research indicates that it has new functions.
  • the present invention also includes compounds, pharmaceutical preparations, analogs and/or salts, hydrates or precursors thereof equivalent to the above compounds I, II or III, IV, V, VI or VII; also including their natural and synthetic Compound.
  • Analogs of the compounds include, but are not limited to, isomers, racemates of the compounds.
  • the compound has one or more asymmetric centers. Therefore, these compounds may exist as racemic mixtures, individual enantiomers, individual diastereomers, diastereomeric mixtures, cis or trans isomers.
  • the “salt” includes, but is not limited to, (1) a salt formed with an inorganic acid such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, etc.; (2) a salt formed with an organic acid such as acetic acid, oxalic acid, or dibutyl Acid, tartaric acid, methanesulfonic acid, maleic acid, or arginine.
  • Other salts include salts formed with alkali metals or alkaline earth metals such as sodium, potassium, calcium or magnesium.
  • precursor of a compound means a compound which, when administered or treated by a suitable method, can be converted into a compound of any of the above compounds in a medium, or an animal, or a human, or A salt or solution of a compound of either compound.
  • GSK3 ⁇ inhibitor (or GSK3 ⁇ inhibitor CHIR99021), TGF ⁇ inhibitor (or TGF ⁇ inhibitor SB431542 or / and A83-01) by weight ratio: (0.046-4.65): (0.038-7.68); Preferably, (0.232-2.325): (0.192-3.84) is present; or in a solution state in a molar ratio: (0.1-10): (0.1-20); preferably, (0.5-5): (0.5- 10) Exist.
  • the small molecule composition is used for chemically inducing direct reprogramming of human tumor cells into non-tumorigenic cells accompanied by apoptosis of tumor cells; however, the composition is biased to induce apoptosis of tumor cells, and the function of transforming tumor cells is weak;
  • the composition further comprises: a retinoid compound: 0.03-6.0 parts by weight; preferably: 0.15-3.0 parts by weight; or a final molar concentration in the solution state: 0.1-20 uM;
  • a retinoid compound 0.03-6.0 parts by weight; preferably: 0.15-3.0 parts by weight; or a final molar concentration in the solution state: 0.1-20 uM;
  • the addition of the above ingredients promotes and enhances tumor cell transformation or apoptosis and expands the type or extent of tumor application.
  • the GSK3 ⁇ inhibitor (or GSK3 ⁇ inhibitor CHIR99021), the TGF ⁇ inhibitor (or the TGF ⁇ inhibitor SB431542 or/and A83-01), and the retinoid compound (or retinoic acid) are weighed.
  • one or two components selected from the group consisting of BMP inhibitor LDN-193189: 0.02-4.65 parts by weight; preferably: 0.203-2.03 parts by weight may also be added.
  • Addition of the above components can further promote or enhance the induction of transdifferentiation or apoptosis of certain extremely malignant tumor cells.
  • the composition is a GSK3 ⁇ inhibitor (such as the GSK3 ⁇ inhibitor CHIR99021), a TGF ⁇ inhibitor (such as the TGF ⁇ inhibitor SB431542 or/and A83-01), a retinoid compound (such as retinoic acid).
  • a GSK3 ⁇ inhibitor such as the GSK3 ⁇ inhibitor CHIR99021
  • a TGF ⁇ inhibitor such as the TGF ⁇ inhibitor SB431542 or/and A83-01
  • a retinoid compound such as retinoic acid
  • BMP inhibitor such as BMP inhibitor LDN-193189
  • BrdU in parts by weight: (0.046-4.65): (0.038-7.68): (0.03-6.0): (0.02-4.65): 0.15-30; Good ground, (0.232-2.325): (0.192-3.84): (0.15-3): (0.203-2.03): (1.5-15) is present; or in the solution state in molar ratio: (0.1-10): (0.1 -20): (0.1-20): (0.05-10): (0.5-100); preferably, (0.5-5): (0.5-10): (0.5-10): (0.5-5): (5-50 )presence.
  • the composition is a GSK3 ⁇ inhibitor (such as the GSK3 ⁇ inhibitor CHIR99021), a TGF ⁇ inhibitor (such as the TGF ⁇ inhibitor SB431542 or/and A83-01), a retinoid compound (such as retinoic acid).
  • a GSK3 ⁇ inhibitor such as the GSK3 ⁇ inhibitor CHIR99021
  • a TGF ⁇ inhibitor such as the TGF ⁇ inhibitor SB431542 or/and A83-01
  • a retinoid compound such as retinoic acid
  • BMP inhibitor such as BMP inhibitor LDN-193189
  • EdU in parts by weight: (0.046-4.65): (0.038-7.68): (0.03-6.0): (0.02-4.65): (0.125-25)
  • (0.232-2.325): (0.192-3.84): (0.15-3): (0.203-2.03): (1.25-12.5) is present; or in a solution state in a molar ratio: (0.1-10): (0.1-20): (0.1-20): (0.05-10): (0.5-100); preferably, (0.5-5): (0.5-10): (0.5-10): (0.5-5 ): (5-50) exists.
  • the dosage form of the composition of the present invention is not particularly limited and may be any dosage form suitable for mammals; the preparation forms include powders, powders, tablets, pills, capsules, sustained release agents, and rate control. Release agents and other solid dosage forms; injections, infusion solutions, suspensions and other liquid dosage forms; and other dosage forms such as gas dosage forms, semi-solid dosage forms, and the like.
  • the dosage form may be, but not limited to, a solid dosage form such as a powder, a granule, a capsule, a sustained release agent, a tablet, or a liquid dosage form such as an injection, an infusion solution, a solution, or a suspension.
  • the preparation method of the composition of the present invention is determined according to the preparation form to be prepared and the administration route, and those skilled in the art can refer to the combination and the ratio provided by the present invention, and then adopt the preparation method of the conventional pharmaceutical composition.
  • the composition of the invention is prepared.
  • the present inventors have for the first time demonstrated that the small molecule compositions of the present invention can be used to develop pharmaceutical or pharmaceutical formulations for the prevention, amelioration or treatment of tumors.
  • the effective dosage of the composition used may vary depending on the mode of administration and the type of tumor to be treated and the severity of the disease. The specific circumstances are determined according to the individual condition of the subject, which is within the range judged by the skilled physician or pharmacist.
  • the tumor or tumor cell includes, but is not limited to, liver cancer, nasopharyngeal carcinoma, lung cancer, gastric cancer, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, osteosarcoma, lymphoma, leukemia, Esophageal cancer, cervical cancer, oral cancer, salivary gland tumor, nasal and paranasal sinus malignant tumor, laryngeal cancer, ear tumor, ocular tumor, thyroid tumor, mediastinal tumor, chest wall, pleural tumor, small intestine tumor, biliary tract tumor, pancreas and Periampullary tumor, mesenteric and retroperitoneal tumor, kidney tumor, adrenal tumor, bladder tumor, testicular tumor, penile cancer, endometrial cancer, ovarian malignant tumor, malignant trophoblastic tumor, vulvar cancer and vaginal cancer, malignant lymphoma, Multiple myeloma, soft tissue tumors, bone tumors, skin and accessory tumors,
  • the invention also provides a medium for inducing direct reprogramming (transdifferentiation) of human tumor cells into non-tumorigenic cells accompanied by apoptosis of tumor cells by small molecule composition (hereinafter referred to as: tumor cell transdifferentiation accompanied by apoptosis medium) ).
  • a specific final concentration of the small molecule composition is selected for formulation.
  • different components in the specific small molecule composition are dissolved in DMSO (dimethyl sulfoxide) or other organic solvent or aqueous solvent according to their different solute properties and different solubility.
  • each small molecule organic solution concentrate reagent is diluted and added to 10% calf serum, 1%
  • the tumor cell transdifferentiation-associated apoptosis medium can be obtained by basal cell culture medium (or serum-free medium containing various cytokines or growth factors) of the streptomycin mixture (100x).
  • the percentage of each component of the medium can also be up and down by 50%; preferably up and down by 30%; more preferably up and down by 20%, such as 10%, 5%; (percentage is in v/v ).
  • the basal cell culture medium includes, but is not limited to, DMEM/F12, MEM, DMEM, F12, IMDM, RPMI1640, Neuronal basal or Fischers, etc., all of which are commercially available.
  • the "serum-free medium” refers to a cell culture medium containing no serum and containing various nutrients (such as growth factors, tissue extracts, etc.) supporting cell proliferation and biological reactions.
  • various nutrients such as growth factors, tissue extracts, etc.
  • An additive such as various cytokines or growth factors other than serum is added to the cell culture medium composed of the basal cell culture medium.
  • the serum-free medium containing various cytokines or growth factors includes, but is not limited to, ITS, N2, B27, etc., all of which are self-preparable or commercially available products.
  • the basal cell culture medium or the serum-free medium is not limited to those exemplified in the present invention.
  • the "tumor cell transdifferentiation accompanying apoptosis medium” is specifically formulated as follows:
  • 1GSK3 ⁇ inhibitor (or GSK3 ⁇ inhibitor CHIR-99021): 0.046-4.65 parts by weight; preferably: 0.232-2.325 parts by weight; or a final concentration of 0.1-10 uM in a solution state; a preferred amount is 0.5 -5uM; and 2TGF ⁇ inhibitor (or TGF ⁇ inhibitor SB431542 or / and A83-01): 0.038-7.68 parts by weight; preferably: 0.192-3.84 parts by weight; or a final concentration of 0.1-20 uM in solution; preferably The amount is: 0.5-10 uM mixed, and the small molecule composition of the present invention for chemically inducing direct reprogramming of human tumor cells into non-tumorigenic cells accompanied by apoptosis of tumor cells is obtained.
  • composition of (1) it may further comprise: a retinoid compound (or retinoic acid): 0.03-6.0 parts by weight; preferably: 0.15-3 parts by weight; or a solution state
  • a retinoid compound or retinoic acid
  • the final concentration is: 0.1-20 uM; preferably 0.5-10 uM; the addition of the above ingredients can promote and enhance the transformation or apoptosis of tumor cells, and expand the applicable tumor type or range.
  • composition of (2) On the basis of the composition of (2), one or two components selected from the group consisting of BMP inhibitor LDN-193189: 0.02-4.65 parts by weight; preferably: 0.203-2.03 may be added. Parts by weight.
  • concentration 0.05-10 uM; preferably 0.5-5 uM; or / and BrdU, or / and EdU: 0.15-30 parts by weight (BrdU), 0.125-25 (EdU); preferably: 1.5-15 parts by weight (BrdU), 1.25-12.5 (EdU); or in solution state (BrdU or / and EdU): final
  • the concentration is: 0.5-100 uM; preferably 5-50 uM.
  • the addition of the above ingredients further promotes or enhances the transformation or apoptosis of certain tumor cells.
  • the present invention also provides an injection or oral administration of an experimental animal for chemically inducing direct reprogramming (transdifferentiation) of a human tumor cell into a non-tumorigenic cell accompanied by apoptosis of the tumor cell.
  • each small molecule composition in any of the above compositions is calculated in terms of kilograms of body weight, and is dissolved in Captisol (1-30%) or Tween-80 (5%). Obtaining the test animal for injection or oral administration in the solution; or taking the corresponding amount of the concentrated reagent of the different components in the composition, and adding it to the physiological saline or phosphate solution for injection (with or without 5% FBS) Laboratory animals for injection or oral administration. It is preferably soluble in Captisol (1-30%).
  • the invention also discloses a method for inducing direct reprogramming (transdifferentiation) of human tumor cells into non-tumorigenic cells accompanied by apoptosis of tumor cells by a small molecule composition, the method steps comprising:
  • Concentrate reagent preparation The composition according to any one of claims 1 to 6, wherein each component is dissolved in an organic solvent or an aqueous solvent to prepare a concentrate reagent; preferably, the organic solvent comprises two Methyl sulfoxide; preferably, the aqueous solvent comprises: water, physiological saline, phosphate buffer;
  • the concentrate reagent in step (1) is separately diluted into a basic cell culture medium containing 5-20% calf serum, 1% streptomycin mixed solution (100x) or contains various cells.
  • a serum-free medium of a factor or a growth factor (such that the concentration of each component conforms to the concentration defined in the composition according to any one of claims 1 to 6), and a medium for inducing tumor cell transdifferentiation with apoptosis is obtained;
  • the percentage of each component of the medium can also be up and down by 50%; preferably 30% up and down; more preferably up and down 20%, such as 10%, 5%.
  • tumor cell transdifferentiation accompanied by apoptosis the tumor cells are suspended in the "tumor cell transdifferentiation accompanying apoptosis medium" prepared in the above step (2), and plated as a treatment group;
  • the solvent such as DMSO or other solvent which dissolves the small molecule composition in the treatment group medium is added in an equal amount to the basal cells containing the mixture of 10% calf serum and 1% streptomycin (100x) used in the same treatment group.
  • the medium or serum-free medium containing various cytokines or growth factors
  • obtain "control medium” percentage is in v/v); then add the same amount of tumor cells as the control group to the "control culture” Suspended in the base, plated, as a control group;
  • the "tumor cell transdifferentiation accompanying apoptotic small molecule composition" of the invention and the prepared medium and reagent thereof and the experimental method thereof can not only develop and prepare a therapeutic tumor drug, but also can be widely used for the prevention and treatment of tumor methods and mechanisms, and clinical Pre-study, pharmacological and toxicological safety tests; non-tumorigenic cells obtained can continue to perform functional tests, tumorigenic multi-target experiments, preclinical studies, etc.
  • This method not only opens up a new field for the prevention and treatment of tumors, but also has broad application prospects; it also enriches the theory of stem cell reprogramming and expands its application range. It has great scientific significance and great application value.
  • the first method of cell reprogramming and chemically induced cell direct reprogramming (transdifferentiation) was applied to tumor treatment research and the expected results were obtained.
  • Small molecules are used to induce transdifferentiation of hepatoma cells into functional (in vitro) non-tumorigenic hepatocytes accompanied by apoptosis of cancer cells; similar ideas and methods have been applied to other tumor cells for transdifferentiation to obtain the same or similar effects.
  • the first method of chemically induced hepatoma cell transdifferentiation was applied to tumor transdifferentiation in vivo (PDX animal model experiment of liver cancer tissue).
  • the results showed that most of the liver cancer tissues in the body were necrotic, and the non-necrotic tissues no longer had the tissue structure of liver cancer, and the cells in them expressed HNF4a, suggesting that they have been transdifferentiated. It is proved that the small molecule composition of the invention has potential therapeutic effect on liver cancer treatment of patients;
  • the applied small molecule property is stable, the time, dosage and combination mode of the action are easy to control, and the effect is stable and reliable;
  • the invention has no foreign gene introduction, does not change the genetic structure of the cell, and avoids the introduction of foreign gene introduction or structural gene change to cause new cancer risk, so it is safe and reliable;
  • the small molecule composition of the present invention is effective not only for inducing transdifferentiation of hepatoma cells with apoptosis, but further studies have shown that induction of nasopharyngeal carcinoma, lung cancer, gastric cancer, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, Transdifferentiation of prostate cancer, osteosarcoma, lymphoma, leukemia and other hematological tumors and substantial tumor cells into non-tumorigenic cells with the same or similar effects with apoptosis;
  • the small molecule composition of the present invention has the potential to be developed into a highly effective and low-toxic new method, a new method, a new drug for controlling or treating liver cancer and other malignant tumors; or to prepare a chemically induced tumor cell to be directly reprogrammed to non-tumorigenicity.
  • the small molecule composition of the present invention and the method for inducing tumor cell transdifferentiation accompanying apoptosis are simple in operation, low in cost, and easy to produce and use.
  • the invention enriches the theory of reprogramming, broadens its application range, opens up a new field of tumor treatment research, and provides new ideas, new methods and new drugs for clinical tumor treatment.
  • Example 1 Tumor cell transdifferentiation accompanied by apoptotic small molecule composition and preparation of its medium and reagent
  • composition or medium is formulated as follows, and can be formulated in a molar concentration or a weight concentration:
  • GSK3 ⁇ inhibitor CHIR-99021 final concentration 2 uM
  • TGF ⁇ inhibitor SB431542 final concentration 5 uM.
  • GSK3 ⁇ inhibitor CHIR-99021 final concentration 3uM
  • TGF ⁇ inhibitor SB431542 final concentration 2 uM.
  • GSK3 ⁇ inhibitor CHIR-99021 final concentration 5 uM
  • TGF ⁇ inhibitor A83-01 final concentration 2 uM.
  • GSK3 ⁇ inhibitor CHIR-99021 final concentration 4 uM
  • the final concentration of TGF ⁇ inhibitor A83-01 was 3 uM
  • Retinoic acid (RA) final concentration 0.5 uM.
  • GSK3 ⁇ inhibitor CHIR-99021 final concentration 3uM
  • TGF ⁇ inhibitor SB431542 final concentration 5 uM
  • Retinoic acid (RA) final concentration 3 uM.
  • TGF ⁇ inhibitor SB431542 final concentration 2 uM
  • Retinoic acid (RA) final concentration 5 uM.
  • GSK3 ⁇ inhibitor CHIR-99021 final concentration 3uM
  • TGF ⁇ inhibitor SB431542 final concentration 1 uM
  • BMP inhibitor LDN-193189 final concentration 0.5 uM.
  • GSK3 ⁇ inhibitor CHIR-99021 final concentration 3uM
  • TGF ⁇ inhibitor SB431542 final concentration 5 uM
  • Retinoic acid (RA) final concentration 3uM
  • BMP inhibitor LDN-193189 0.5 uM.
  • GSK3 ⁇ inhibitor CHIR-99021 final concentration 3uM
  • TGF ⁇ inhibitor SB431542 final concentration 2 uM
  • BMP inhibitor LDN-193189 final concentration 0.5 uM.
  • GSK3 ⁇ inhibitor CHIR-99021 final concentration 3uM
  • TGF ⁇ inhibitor SB431542 final concentration 5 uM
  • BMP inhibitor LDN-193189 2 uM.
  • GSK3 ⁇ inhibitor CHIR-99021 final concentration 3uM
  • TGF ⁇ inhibitor SB431542 final concentration 7.5 uM
  • BMP inhibitor LDN-193189 final concentration 0.5 uM.
  • GSK3 ⁇ inhibitor CHIR-99021 final concentration 5 uM
  • TGF ⁇ inhibitor SB431542 final concentration 2 uM
  • Retinoic acid (RA) final concentration 5 uM.
  • GSK3 ⁇ inhibitor CHIR-99021 final concentration 4 uM
  • the final concentration of TGF ⁇ inhibitor A83-01 was 3 uM
  • GSK3 ⁇ inhibitor CHIR-99021 final concentration 3uM
  • TGF ⁇ inhibitor SB431542 final concentration 5 uM
  • Retinoic acid (RA) final concentration 3uM
  • BMP inhibitor LDN-193189 0.5 uM
  • GSK3 ⁇ inhibitor BIO final concentration 3uM
  • TGF ⁇ inhibitor SB431542 final concentration 7.5 uM
  • BMP inhibitor LDN-193189 final concentration 0.5 uM.
  • GSK3 ⁇ inhibitor CHIR-99021 final concentration 5 uM
  • TGF ⁇ inhibitor LY2109761 final concentration 2 uM
  • Retinoic acid (RA) final concentration 5 uM.
  • GSK3 ⁇ inhibitor CHIR-99021 final concentration 3uM
  • TGF ⁇ inhibitor RepSox final concentration 7.5 uM
  • BMP inhibitor LDN-193189 final concentration 0.5 uM.
  • TGF ⁇ inhibitor A83-01 final concentration 5 uM
  • BMP inhibitor LDN-193189 2 uM.
  • GSK3 ⁇ inhibitor CHIR-99021 final concentration 4 uM
  • TGF ⁇ inhibitor SB431542 final concentration 3 uM
  • 9-cis retinoic acid final concentration 0.5 uM.
  • GSK3 ⁇ inhibitor TWS119 final concentration 4 uM
  • TGF ⁇ inhibitor A83-01 final concentration 3 uM
  • Retinoic acid (RA) final concentration 0.5 uM.
  • Each specific small molecule composition is first dissolved in DMSO according to the aforementioned "culture method" step (1) to prepare a concentrate reagent.
  • Tumor cell transdifferentiation is accompanied by apoptotic medium preparation
  • the tumor cells prepared in the above experimental step 1 are transdifferentiated and the DMSO concentrate reagents of the components 1 to 20 of the apoptotic compositions 1 to 20 are prepared according to the above-mentioned culture method step (2) (the selected basic medium is DMEM/F12), and the tumor cells are obtained.
  • the differentiation is accompanied by the apoptosis medium 1-20 (ie, the final concentration of the compound of the medium 1 and the composition 1 is the same, the final concentration of the compound of the medium 2 and the composition 2 is the same, ..., the final concentration of the compound of the medium 20 and the composition 20 the same).
  • Tumor cell transdifferentiation with apoptosis composition 21 was prepared in terms of mg/kg (body weight):
  • TGF ⁇ inhibitor SB431542 0.5 mg/kg
  • Retinoic acid 0.2 mg/kg
  • BMP inhibitor LDN-193189 0.2 mg / kg
  • Example 2 Hepatoma cell SMMC-7721 was induced to undergo transdifferentiation with apoptosis by medium 6
  • Tumor cell transdifferentiation is accompanied by apoptosis culture
  • liver cancer cells SMMC-7721 were suspended in the above-prepared medium 6, and plated as a treatment group.
  • DMSO control culture
  • basal cell culture medium DMEM/F12 containing 10% calf serum and 1% streptomycin mixture (100x). Base (the percentages are in v/v); then the same amount of liver cancer cells SMMC-7721 as the treatment group were added to the DMSO "control medium", and plated, as a control group;
  • Subculture step discard the original culture solution, wash once with PBS, add cells to digest the cells, digest the cells at 37 ° C for 1-5 minutes, terminate the cell digestion, centrifuge, discard the supernatant, and resuspend the cell pellet according to 1:1-1 : 3 passaging paving. Incubate according to the first and second steps of the experimental procedure, and change the solution once every 2-4 days.
  • the digestive juice used is trypsin (also available as EDTA, Acutase, TrypleE) and the like. 3-7 days pass once.
  • the transdifferentiation of the experimental steps 1 and 2 was accompanied by apoptosis culture and subculture of the hepatoma cells for 1-3 weeks, and after the apoptotic cells were removed by centrifugation, the non-apoptotic hepatoma cells were transdifferentiated into non-tumorigenic hepatocyte-like cells; Non-tumorigenic liver Cell-like cells can be used for other scientific experiments.
  • liver cancer cell SMMC-7721 was induced to transdifferentiate, completely altered morphology, showing that it has been transdifferentiated;
  • B picture liver cancer cell SMMC-7721 was induced to transdifferentiate with apoptosis statistics. There were different degrees of early and late apoptosis in the treatment group at different treatment time periods. The control group had almost no early apoptosis and some advanced natural apoptosis.
  • Example 3 small molecule combination (medium 1, 4) induced hepatoma cell HepG2 transdifferentiation with apoptosis
  • the treatment group and the control group setting and the culture experiment procedure were the same as those in Example 2 except that the medium 6 was replaced with the mediums 1 and 4, respectively.
  • Fig. 2 The experimental results of the culture treatment for 1-3 weeks are shown in Fig. 2.
  • Fig. A HepG2 cells were induced to transdifferentiate by (medium 4), and the morphology was completely changed, indicating that they had been transdifferentiated; except for the hepatoma cells which were induced to apoptosis in the early and late stages, the remaining liver cancer cells were all transdifferentiated.
  • B hepatocellular carcinoma cell HepG2 was induced by (medium 1) apoptosis; the treatment group had different degrees of early and late apoptosis in different treatment periods, and the control group had almost no early apoptosis, and there was some late natural apoptosis. .
  • Example 4 5-Fu-resistant liver cancer cells 7402/5-Fu were induced to be transdifferentiated and accompanied by apoptosis in medium 5 and 2, respectively.
  • the treatment group and the control group setting and the culture experiment procedure are the same as those in Example 2, except that the tumor cell transdifferentiation is accompanied by the apoptosis medium 5 or the tumor cell transdifferentiation is accompanied by the apoptosis medium 2 to replace the tumor cell transdifferentiation with the apoptosis medium. 6.
  • liver cancer cell 7402/5-Fu was induced to transdifferentiate into hepatocyte-like cells by (medium 5), and the morphology was completely changed, indicating that it had been transdifferentiated;
  • liver cancer cells 7402/5-Fu were (medium 2 Induction of transdifferentiation is accompanied by apoptosis statistics. There were different degrees of early and late apoptosis in the treatment group at different treatment time periods. There was no early apoptosis in the control group, and there was very little late natural apoptosis.
  • liver cancer cells SMMC-7721, HepG2, 7402/5-Fu were induced to be transdifferentiated into hepatocyte-like cells with normal hepatocyte function by medium 6, 4, and 5, respectively.
  • Treatment group and control group settings and culture experiment procedures were the same as in Examples 2, 3 and 4, and tumor cell transdifferentiation was used with apoptosis medium 6, 4, 5. After the apoptotic cells were removed by centrifugation in the treatment group treated for 2 weeks, the non-apoptotic liver cancer cells were transdifferentiated into non-tumorigenic hepatocyte-like cells. The control group was cultured for the same time to collect the cells.
  • Hepatocyte-like cells SMMC-7721 (medium 6), HepG2 (medium 4), and 7402/5-Fu (medium 5) were induced to transdifferentiate, respectively, and the obtained hepatocyte-like cells had normal hepatocyte function.
  • PAS glycogen staining
  • Oil-red oil red staining, reflecting the fat intake function.
  • hepatocyte-like cells obtained after transdifferentiation of liver cancer cells as described above have normal human hepatocyte-related functions.
  • Example 6 Hepatoma cells SMMC-7721, HepG2, 7402/5-Fu were induced to be transdifferentiated into cells with hepatocyte function by medium 10, 11, and 12, respectively.
  • the treatment group and the control group were set up and the culture experiment procedure was the same as in Example 2, and the tumor cell transdifferentiation was accompanied by the apoptosis medium 10, 11, and 12. After the apoptotic cells were removed by centrifugation in the treatment group treated for 2 weeks, the cell culture supernatants and cells of the treatment group and the control group were collected.
  • albumin secretion and urea production functions of the cells cultured in the treatment group and the control group were separately detected by the ELISA method using a kit. See the kit instructions for specific experimental procedures.
  • Albumin test kit Bioassay System/DIAG-250, BCG Albumin assay kit
  • urea test kit Bioassay System/DIUR-500, Urea assay kit.
  • rifampicin induced CYP3A4
  • omeprazole induced CYP1A2
  • Different concentrations of rifampicin (1uM, 10uM, 25uM) or omeprazole (1uM, 10uM, 25uM) were added to the cell culture medium of the treatment group and the control group for 48 hours, then the cellular RNA was collected and qRT-PCR was used.
  • CYP3A4 and CYP1A2 gene expression levels were quantified by cells of different treatment conditions.
  • liver cancer cell SMMC-7721 was induced to transdifferentiate (medium 10), and the obtained hepatocyte-like cells had normal hepatocyte function;
  • Hepatoma cell HepG2 was induced to transdifferentiate by (medium 11), and the obtained hepatocyte-like cells had normal hepatocyte function;
  • liver cancer cells 7402/5-Fu were induced to transdifferentiate (medium 12), and the obtained hepatocyte-like cells have normal hepatocyte function;
  • the blue column represents early apoptosis
  • the red column represents late apoptosis
  • T1W, T2W, T3W represent treatment for 1, 2, 3 weeks, respectively
  • Rif rifampicin
  • Ome Omelapola.
  • the hepatocyte-like cells obtained by transdifferentiation have normal human hepatocyte-related functions, and the transdifferentiated hepatoma cells obtain normal hepatocyte albumin secretion (ALB), urea production (Urea), CYP1A2 induction and CYP3A4 induction function.
  • ALB hepatocyte albumin secretion
  • Urea urea production
  • CYP1A2 induction CYP3A4 induction function.
  • liver cancer cells SMMC-7721, HepG2 and 7402/5-Fu were respectively (medium 6, 4, 5) Inducing transdifferentiation, the obtained hepatocyte-like cells are no longer tumorigenic in vitro or in vivo.
  • the treatment group and the control group were set up and the culture experiment procedure was the same as in Example 2, and the tumor cell transdifferentiation was carried out with the apoptosis medium 6, 4, 5 for 2 weeks, and the apoptotic cells were removed after the treatment group was removed. It was transdifferentiated into hepatocyte-like cells; the obtained hepatocyte-like cells were not tumorigenic in vitro or in vivo.
  • Method steps 1 ⁇ 10 3 cells of the treatment group and the control group were planted in a 6-well plate, and cultured in the same manner as in Example 2, cultured for 2 weeks; crystal violet staining, taking photos, observing and counting cell growth to form clones.
  • Panel A Hepatocyte-like cells SMMC-7721 (medium 6 treatment), HepG2 (medium 4 treatment) and 7402/5-Fu (media 5 treatment) were not formed into clones of hepatocyte-like cells obtained after transdifferentiation. No tumorigenicity in vitro.
  • Hepatocyte-like cells obtained by transdifferentiation of liver cancer cells for 20 days were digested into single cells by digestive juice, centrifuged, rinsed with PBS, counted in cells, and 1 ⁇ 10 6 cells were taken and injected into the hind legs of 5-week-old nude mice.
  • Subcutaneously (right), cancer cells treated with DMSO "control medium" for the same time were injected as a control into the hind legs of the same nude mouse (left side), and the experiment was terminated 4 weeks, and the tumor was isolated and photographed.
  • Panel B Liver cancer cell SMMC-7721 was induced to transdifferentiate by medium 6, and the obtained hepatocyte-like cells did not produce tumors in vivo and lost tumorigenicity.
  • Figure C 7402/5-Fu was induced to transdifferentiate by medium 8, and the obtained hepatocyte-like cells did not produce tumors in vivo and lost tumorigenicity; C map showed that no tumor was formed in the right-hand side of the nude mice; Tumor formation; C picture The figure below shows the appearance of the tumor anatomical shape formed by the control group.
  • Example 8 PDX animal model test of patient liver cancer tissue (transdifferentiation accompanied by apoptotic small molecule composition 8)
  • Tumor tissue surgically removed from liver cancer patients was implanted into a subcutaneous tumor of 5 weeks old nude mice (PDX model).
  • the tumors of the nude mice were isolated and subcutaneously implanted into nude mice of 5 weeks old.
  • the drug is treated when the tumor grows to a size of 5-10 mm.
  • Tumor cells prepared in Example 1 were transdifferentiated with apoptosis.
  • Experimental animals were injected with Reagent 8 for injection, 3 times/week, and injected with DMSO saline injection as a control. The experiment was terminated after 4 weeks.
  • the tumor was isolated, photographed, fixed tumor tissue, and HE stained.
  • Example 9 Normal human fibroblasts and hepatocytes were transdifferentiated by tumor cells and treated with apoptosis medium 8 and 3, respectively.
  • the treatment group and the control group were set up and the experimental culture method was the same as in Example 2, except that the normal human fibroblasts and hepatocytes were cultured in turn with tumor cell transdifferentiation with apoptosis medium 8 or 3.
  • Example 10 nasopharyngeal carcinoma cell line HNE and lung cancer cell H460 were induced to transdifferentiate with media 9, 7 and accompanied by apoptosis
  • the treatment group and the control group setting and the culture experiment procedure were the same as those in Example 2.
  • the experimental results of the culture treatment for 2 weeks are shown in Fig. 9.
  • nasopharyngeal carcinoma cell line HNE medium 9 treatment
  • the morphology of the cancer cells was completely changed, indicating that it had been transdifferentiated; the treated group lung cancer cells H460 were treated separately (media 9, 7), and the lung cancer cells H460 were treated. (Medium 7) almost exclusively induced apoptosis; whereas the control lung cancer cell H460 showed almost no apoptosis.
  • Example 11 gastric cancer cells SGC-7901 and MKN28 were induced to transdifferentiate (with media 12, 13) and accompanied by apoptosis, respectively.
  • the treatment group and the control group setting and the culture experiment procedure were the same as those in Example 2.
  • Fig. 10 The results of the culture treatment for 2 weeks are shown in Fig. 10. After the transdifferentiation of the gastric cancer cells SGC-7901 (medium 12) and MKN28 (medium 13) in the treated group, the cell morphology was completely changed, indicating that it had been transdifferentiated.
  • pancreatic cancer cell SW1990 was induced to transdifferentiate by medium 12
  • the treatment group and the control group setting and the culture experiment procedure were the same as those in Example 2.
  • pancreatic cancer cell SW1990 treated with (medium 12) for 2 weeks are shown in Fig. 11. After the transdifferentiation of the cancer cells in the treated group, the morphology completely changed, indicating that it has been transdifferentiated.
  • Example 13 breast cancer cell SKBr3 was induced to transdifferentiate by medium 13
  • the treatment group and the control group setting and the culture experiment procedure were the same as those in Example 2.
  • the results of the culture treatment for 2 weeks (medium 13) are shown in Fig. 12.
  • the morphology completely changed, indicating that it has been transdifferentiated.
  • Example 14 leukemia cell U937, B cell lymphoma SUDHL-4 was induced to transdifferentiate with apoptosis by medium 10, 11
  • the treatment group and the control group setting and the culture experiment procedure were the same as those in Example 2.
  • the results of the culture treatment for 2 weeks are shown in Fig. 13, and the leukemia cells U937 (culture medium 10 culture) and B cell lymphoma cells SUDHL-4 (culture medium 11 culture) of the treatment group were largely apoptotic.
  • Example 15 breast cancer cell SKBr3 and gastric cancer cell MKN28 were induced to be transdifferentiated by medium 13, 14
  • the treatment group and the control group setting and the culture experiment procedure were the same as those in Example 2.
  • the results of the culture treatment for 2 weeks are shown in Fig. 14. After transdifferentiation of the breast cancer cells SKBr3 (medium 13) and gastric cancer cells MKN28 (medium 14), the cell morphology was completely changed, indicating that it had been transdifferentiated.
  • Example 16 Intestinal cancer cell HCT116 was induced to transdifferentiate into non-tumorigenic cells by medium 9.
  • the treatment group and the control group setting and the culture experiment procedure were the same as those in Example 2.
  • the results of the culture treatment for 2 weeks are shown in Fig. 15.
  • the right panel shows that after transdifferentiation of the intestinal cancer cell HCT116 (medium 9) in the treated group, the cells no longer form clones and lose tumorigenicity.
  • Example 17 prostate cancer cell line PC-3, ovarian cancer cells SKOV3 and A2780 were induced to lose tumorigenicity after induction of transdifferentiation by medium 5, 7, and 8, respectively.
  • the treatment group and the control group setting and the culture experiment procedure were the same as those in Example 7. After 2 weeks of treatment, the experimental results are shown in Figures 16 and 17.
  • the right panel of Fig. 16 and the upper and lower panels of Fig. 17 respectively show the prostate cancer PC-3 (medium 5) in the treated group, and the ovarian cancer cells SKOV3 (medium 7) and A2780 (medium 8) were no longer formed after induced differentiation. Cloning, loss of tumorigenicity in vitro.
  • Example 18 gastric cancer cell MKN28, breast cancer cell SKbr3 was lost to tumorigenicity after induction of transdifferentiation by medium 10 and 11, respectively.
  • the treatment group and the control group setting and the culture experiment procedure were the same as those in Example 7. After 2 weeks of treatment, the experimental results are shown in Fig. 18.
  • the upper right panel of Fig. 18 shows that the gastric cancer cells MKN28 (media 10 treatment) and breast cancer cells SKbr3 (media 11 treatment) in the treatment group no longer form clones after transdifferentiation, and the tumorigenicity in vitro is lost.
  • Example 19 glioma cells T98G, U87MG were induced to transdifferentiate with media 4, 5 and accompanied by apoptosis.
  • the treatment group and the control group setting and the culture experiment procedure were the same as those in Example 14, and the results of the culture treatment for 2 weeks are shown in Fig. 19.
  • the glioma cells T98G (medium 4 treatment) and U87MG (media 5 treatment) were induced to undergo complete differentiation after transdifferentiation, indicating that they had been transdifferentiated.
  • Example 20 Patient liver cancer tissue PDX animal model test (tumor cell transdifferentiation accompanied by apoptotic small molecule composition 15)
  • the experimental animal injection reagent, the treatment group and the control group were set up and the experimental procedure was the same as in Example 8, except that the injection reagent 15 prepared by the tumor cell transdifferentiation accompanied with the apoptosis composition 15 was used, and the experimental results are shown in Fig. 20.
  • the liver cancer tissues and cells of the treatment group were treated with the reagent 15 for injection for 3 weeks, and a large area was necrotic, and the cancer tissue structure was destroyed or lost.
  • the treatment group on the right shows that after treatment with small molecule reagent for 3 weeks, residual tissue and cells express HNF4a, a unique marker of human hepatocytes, suggesting that transdifferentiation has occurred.
  • Example 21 lung cancer cells A549, H1299 and H460 were induced to be transdifferentiated by medium 14, 13, 9 respectively, and no longer tumorigenic in vivo or in vitro.
  • Panel A Results of culture treatment for 2 weeks.
  • the treatment group of the following figure showed that lung cancer cells A549 (medium 14), H1299 (medium 13 treatment) and H460 (media 9 treatment) were induced to transdifferentiate, respectively, and clones were no longer formed. Loss of tumorigenicity in vitro;
  • Example 22 lung cancer cell H1299 was induced to transdifferentiate into non-tumorigenic cells by medium 15
  • the treatment group and the control group setting and the culture experiment procedure were the same as those in Example 2.
  • Fig. 22 The results of the culture treatment for 2 weeks are shown in Fig. 22, and the right panel shows that the lung cancer cells H1299 cells (treated with medium 15) in the treated group no longer form clones after transdifferentiation, and the tumorigenicity is lost.
  • Example 23 ovarian cancer cells A2780, SKOV3 were induced to transdifferentiate into non-tumorigenic cells, respectively (medium 16, 17)
  • the treatment group and the control group setting and the culture experiment procedure were the same as those in Example 2.
  • Fig. 23 The results of the culture treatment for 2 weeks are shown in Fig. 23.
  • the upper and lower panels respectively show that the ovarian cancer cells A2780 (treated with medium 16) and SKOV3 (treated with medium 17) in the treated group no longer form clones after transdifferentiation, and the tumorigenicity is lost.
  • Small molecule compositions consisting of other small molecules of LY2109761 or RepSox and GSK3 ⁇ inhibitors and retinoic acid compounds or BMP inhibitors, which induce TGF ⁇ inhibitors, also have the same effect of inducing tumor cell transdifferentiation with apoptosis.
  • Example 24 prostate cancer cell PC9 was induced to transdifferentiate (medium 18)
  • the small molecule composition of other small molecule CHIR-98014 and TGF ⁇ inhibitor and BMP inhibitor indicating GSK3 ⁇ inhibitor also has the same effect of inducing tumor cell transdifferentiation with apoptosis.
  • Example 25 gastric cancer SGC-7901 cells were induced to undergo transdifferentiation and accompany apoptosis by medium 19 and 20, respectively.
  • Gastric cancer SGC-7901 cells lose their tumorigenicity after being induced to transdifferentiate by medium 19.
  • the small molecule composition of other small molecule 9-cis retinoic acid and a GSK3 ⁇ inhibitor and a TGF ⁇ inhibitor indicating a retinoid compound also has the same effect of inducing tumor cell transdifferentiation with apoptosis.
  • the treatment group and the control group setting and the culture experiment procedure were the same as those in Example 2.
  • Panel B There were different degrees of early and late apoptosis in the treatment group at different treatment periods, and there were few early and late natural apoptosis in the control group. The apoptosis values of the control group and the treatment group were statistically different (p ⁇ 0.005).
  • TWS119, TGF ⁇ inhibitor and retinoids which are other small molecules of GSK3 ⁇ inhibitor, also has the same effect of inducing tumor cell transdifferentiation with apoptosis.
  • Example 26 Pancreatic cancer SW1990 cells lose tumorigenicity after induction of transdifferentiation by medium 16
  • the treatment group and the control group setting and the culture experiment procedure were the same as those in Example 2. After 2 weeks of treatment, the experimental results are shown in Fig. 26.
  • the pancreatic cancer SW1990 cells (treated with medium 16) in the treatment group on the right were no longer formed into clones after transdifferentiation, and the tumorigenicity was lost in vitro.
  • the small molecule composition of LY2109761 and GSK3 ⁇ inhibitor and retinoids, which are other small molecules of TGF ⁇ inhibitor, also have the same effect of inducing tumor cell transdifferentiation with apoptosis.

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Abstract

一种基于化学诱导细胞直接重编程机理，诱导人肿瘤细胞直接重编程(转分化)为非致瘤性细胞并伴随肿瘤细胞凋亡的方法及其小分子组合物，以及该小分子组合物制备的培养基和试剂。

Description

小分子组合物诱导人肿瘤细胞直接重编程为非致瘤性细胞的方法 技术领域
本发明属于肿瘤学、干细胞重编程、药学交叉领域；更具体地，本发明涉及一种利用小分子组合物多靶点诱导人肿瘤细胞直接重编程(转分化)为非致瘤性细胞并伴随肿瘤细胞凋亡的方法及其小分子组合物；该小分子组合物可添加药物载体或赋型剂研发制备成临床***的药物或药物配方；或添加水性溶剂或有机溶剂、基础培养基或无血清培养基制备成试剂或培养基。
背景技术
据世卫组织报告，2012年全球约有1.41千万新发癌症病例，820万患者死于癌症(CA期刊《2012全球癌症统计》)；而2011年中国癌症发病人数为337万，死亡211万；每分钟有6.4人被诊断癌症，有5人死于癌症；死亡率高低排名依次为：肺癌、肝癌、胃癌、食管癌和结直肠癌；五年存活率最低是肝癌10.1％，其次是肺癌16.1％(《2015中国肿瘤登记年报》)。因此，目前肿瘤呈现发病率逐年增高趋势。
肿瘤是由处于各种不同分化程度的异常细胞组成，且具异质性特点。现有放射治疗(放疗)、化学药物治疗(化疗)、靶向治疗、生物免疫治疗、诱导分化后杀灭等肿瘤治疗方法，均是针对如何杀灭肿瘤细胞，临床实践证明其无法克服肿瘤异质性，且疗效有限、毒副作用大、易产生耐药物、复发率高、五年存活率低。因此,本领域迫切需要开发新型的高效低毒抗肿瘤药物，以期为临床抗肿瘤治疗、提高患者存活率提供新的途径。
本发明的创新思路是：将杀灭肿瘤细胞改为直接转分化其为正常或非致瘤性细胞，从而开创一种能克服肿瘤异质性,且高效低毒的新治疗方法。
细胞重编程(Cell reprogramming)是通过调控细胞信号通路及表观遗传(epigenetics)修饰的变化，使细胞从一种类型向另外一种类型转化(本发明所指细胞重编程为诱导细胞重编程)。细胞重编程包括诱导多能细胞(iPSCs)重编程和细胞直接重编程(转分化)，已广泛应用于正常细胞类型的转化。亦有报道通过外源转录因子将个别肿瘤细胞重编程为iPSCs(仍保有其致瘤性)。本发明不用转录因子，仅用化学小分子诱导调控肿瘤细胞直接重编程(转分化)为非致瘤性细胞并伴随肿瘤细胞凋亡的方法，则未见任何文献报道。
发明内容
本发明的目的在于提供一种多靶点诱导人肿瘤细胞直接重编程(转分化)为非致瘤性细胞并伴随肿瘤细胞凋亡的小分子组合物(以下简称：肿瘤细胞转分化伴随凋亡小分子组合物)及其方法；该小分子组合物可添加药物载体或赋型剂开发制备成临床治疗肿 瘤的药物或药物配方；或添加水性溶剂或有机溶剂、基础培养基或无血清培养基制备成科研用试剂或培养基。
本发明已基本明确的机理，主要是通过GSK3β抑制剂、TGFβ抑制剂两类小分子组合，多靶点诱导肿瘤细胞的GSK3β、TGFβ信号通路改变，以及维甲酸类或诱导表观遗传变化的化合物的协同作用，继而调控肿瘤细胞表观遗传的改变，重编肿瘤细胞的基因表达谱，从而将肿瘤细胞转分化为非致瘤性细胞；而不能转分化的肿瘤细胞则伴随凋亡。对某些种类的肿瘤细胞，还可添加BMP抑制剂诱导调控BMP信号通路，或添加BrdU，或EdU，会有较好效果。需要说明的是，GSK3β抑制剂、TGFβ抑制剂包含了两种类别，功能相同，或诱导靶点相同的系列小分子，所形成的不同组合，都能够不同程度地诱导肿瘤细胞转分化，并伴随不同程度的肿瘤细胞凋亡。BMP抑制剂、维甲酸类化合物亦是类似情况。因此，其功能相同或诱导靶点相同，对同一条信号通路起相同效应的同类小分子化合物，以及所构成的能够诱导调控肿瘤细胞直接转分化为非致瘤性细胞的小分子组合都属于本发明保护范围内。
“致瘤性”为肿瘤细胞的共性，本发明成功地将异质性最强的肝癌细胞转分化为非致瘤性细胞，以此为突破点，相继将胰腺癌、肺癌、胃癌、乳腺癌、淋巴癌、神经胶质瘤等为代表的各种肿瘤或肿瘤细胞，不同程度地化学诱导转分化为非致瘤性细胞，并伴随不同程度的肿瘤细胞凋亡。因此本发明的“小分子组合物诱导人肿瘤细胞直接重编程(转分化)为非致瘤性细胞并伴随肿瘤细胞凋亡的方法及小分子组合物”，所针对的对象，是以肝癌为代表的各种肿瘤或其细胞，即能够将各种肿瘤细胞都不同程度的转分化为非致瘤性细胞，并伴随肿瘤细胞不同程度的凋亡。
在本发明的第一方面，提供一种用于化学诱导人肿瘤细胞直接重编程转化或转分化为非致瘤性细胞并伴随肿瘤细胞不同程度凋亡的小分子组合物(或配方)，所述的组合物包括GSK3β抑制剂，TGFβ抑制剂；或所述的组合物由GSK3β抑制剂，TGFβ抑制剂组成。
在一个优选例中，所述的组合物包括：
GSK3β抑制剂：0.046-4.65重量份；和
TGFβ抑制剂：0.038-7.68重量份。
在另一优选例中，所述的组合物可为溶液状态组合物，包括：
GSK3β抑制剂：终浓度为：0.1-10uM；和
TGFβ抑制剂：终浓度为：0.1-20uM。
在另一优选例中，所述的组合物包括：
GSK3β抑制剂：0.232-2.325重量份；和
TGFβ抑制剂：0.192-3.84重量份。
在另一优选例中，所述的组合物可为溶液状态组合物，包括：
GSK3β抑制剂：终浓度为：0.5-5uM；和
TGFβ抑制剂：终浓度为：0.5-10uM。
在另一优选例中，GSK3β抑制剂，TGFβ抑制剂相加的重量占组合物总重量的0.01～99.9％；例如为0.1～50％(如在溶液状态下)，或50～99.9％。更具体地，如1％，5％，10％，20％，30％等。
在另一优选例中，所述的组合物中，GSK3β抑制剂(或GSK3β抑制剂CHIR99021)、TGFβ抑制剂(或TGFβ抑制剂SB431542或/和A83-01)重量份比为：(0.046-4.65)﹕(0.038-7.68)；较佳地，(0.232-2.325)﹕(0.192-3.84)；或溶液状态下摩尔比为：(0.1-10)﹕(0.1-20)；较佳地，(0.5-5)﹕(0.5-10)。
在另一优选例中，所述组合物还包括：
维甲酸类化合物：0.03-6.0重量份；较佳地为0.15-3重量份。添加维甲酸类化合物，可以促进和增强肿瘤细胞转分化并伴随凋亡，或扩大适用肿瘤类型或范围。
在另一优选例中，所述的组合物可为溶液状态组合物，维甲酸类化合物终浓度为：0.1-20uM；较佳地为0.5-10uM。
在另一优选例中，GSK3β抑制剂，TGFβ抑制剂和维甲酸类化合物相加的重量占组合物总重量的0.02～99.9％；例如为0.2～50％或50～99.9％。更具体地，如1％，5％，10％，20％，30％，40％等。
在另一优选例中，所述的组合物中，GSK3β抑制剂(或GSK3β抑制剂CHIR99021)，TGFβ抑制剂(或TGFβ抑制剂SB431542或/和A83-01)和维甲酸类化合物(或维甲酸)以重量份比：(0.046-4.65)﹕(0.038-7.68)﹕(0.03-6.0)；较佳地，以(0.232-2.325)﹕(0.192-3.84)﹕(0.15-3)存在；或以溶液状态下摩尔比：(0.1-10)﹕(0.1-20)：(0.1-20)；较佳地，(0.5-5)﹕(0.5-10)﹕(0.5-10)存在。
在另一优选例中，所述组合物还可添加包括选自下组的一种或多种成分：
BMP抑制剂(如LDN-193189)：0.02-4.65重量份；较佳地为：0.203-2.03重量份；或
BrdU：0.15-30重量份；较佳地1.5-15重量份；或
EdU：0.125-25重量份；较佳地1.25-12.5重量份。
在另一优选例中，所述的组合物可为溶液状态的组合物，包括选自下组的一种或多种成分：
BMP抑制剂终浓度为：0.05-10uM；较佳地为0.5-5uM；
BrdU终浓度为：0.5-100uM；较佳地为5-50uM；或
EdU终浓度为：0.5-100uM；较佳地为5-50uM。
添加BMP抑制剂(如LDN-193189)、BrdU或EdU，可进一步促进或增强某些恶性肿瘤细胞转分化并伴随凋亡。
在另一优选例中，GSK3β抑制剂，TGFβ抑制剂，维甲酸类化合物，和/或BMP抑制剂，和/或BrdU(或/和EdU)相加的重量占组合物总重量的0.02～99.9％；例如为0.2～ 50％或50～99.9％。更具体地，如1％，5％，10％，20％，30％，40％等。
在另一优选例中，所述的组合物中，GSK3β抑制剂(如GSK3β抑制剂CHIR99021)，TGFβ抑制剂(如TGFβ抑制剂SB431542或/和A83-01)，维甲酸类化合物(如维甲酸)，BMP抑制剂(如BMP抑制剂LDN-193189)和BrdU以重量份比：(0.046-4.65)﹕(0.038-7.68)﹕(0.03-6.0)﹕(0.02-4.65)﹕0.15-30；较佳地，(0.232-2.325)﹕(0.192-3.84)﹕(0.15-3)﹕(0.203-2.03)﹕(1.5-15)存在；或溶液状态下以摩尔比：(0.1-10)﹕(0.1-20)﹕(0.1-20)﹕(0.05-10)﹕(0.5-100)；较佳地，(0.5-5)﹕(0.5-10)﹕(0.5-10)﹕(0.5-5)﹕(5-50)存在。
在另一优选例中，所述的组合物中，GSK3β抑制剂(如GSK3β抑制剂CHIR99021)，TGFβ抑制剂(如TGFβ抑制剂SB431542或/和A83-01)，维甲酸类化合物(如维甲酸)，BMP抑制剂(如BMP抑制剂LDN-193189)和EdU以重量份比：(0.046-4.65)﹕(0.038-7.68)﹕(0.03-6.0)﹕(0.02-4.65)﹕(0.125-25)；较佳地，(0.232-2.325)﹕(0.192-3.84)﹕(0.15-3)﹕(0.203-2.03)﹕(1.25-12.5)存在；或溶液状态下以摩尔比：(0.1-10)﹕(0.1-20)﹕(0.1-20)﹕(0.05-10)﹕(0.5-100)；较佳地，(0.5-5)﹕(0.5-10)﹕(0.5-10)﹕(0.5-5)﹕(5-50)存在。
上述重量份比的重量单位可以是：千克(kg)、毫克(mg)、微克(ug)等任一重量单位；摩尔浓度比的摩尔单位可以是：摩(M)、毫摩(mM)、微摩(uM)等任一摩尔浓度单位。
所述的组合物应用于大动物和肿瘤病人时，按小动物使用剂量通过相应的专业换算公式，换算出大动物或人的有效使用剂量(包括固态或溶液态的剂量换算)，也属于本发明的保护范围。
在另一优选例中，所述的GSK3β抑制剂包括但不限于：CHIR-99021、BIO、IM-12、TWS119、1-Azakenpaullone、CHIR-98014、Tideglusib、AR-A014418、LY2090314、SB216763、AZD1080等为代表的，具相同功能，或诱导相同靶点的同一类GSK3β信号通路抑制剂或化合物，或与它们等效的药剂制品、类似物、异构体和/或其盐、水合物或前体，或其组合；较佳地为GSK3β抑制剂CHIR-99021。
所述的TGFβ抑制剂包括但不限于：SB431542、A83-01、SB525334、LY2109761，RepSox、SD-208、GW788388、SB505124、EW-7197等为代表的，具相同功能，或诱导相同靶点的同一类TGFβ信号通路抑制剂或化合物，或与它们等效的药剂制品、类似物、异构体和/或其盐、水合物或前体，或其组合；较佳地为TGFβ抑制剂SB431542或/和A83-01。
所述的维甲酸类化合物是天然或人工合成的，包括但不限于：维甲酸(别名：全反式维甲酸，ATRA)、13-顺式维甲酸、9-順式维甲酸、异维甲酸等为代表的，具相同功能，或诱导相同靶点的同一类维甲酸类诱导分化剂或化合物，或与它们等效的药剂制品、类似物、异构体和/或其盐、水合物或前体，或其组合；较佳地为维甲酸(Retinoic acid，RA)。
所述的BMP抑制剂包括但不限于：LDN-193189、K02288、DMH1等为代表的，具相同功能，或诱导相同靶点的同一类BMP信号通路抑制剂或化合物，或与它们等效的药剂制品、类似物、异构体和/或其盐、水合物或前体，或其组合；较佳地为BMP抑制剂LDN-193189。
在另一优选例中，所述的组合物是药物组合物，还包含药学上可接受的载体或赋形剂，其载体或赋形剂包括(但不限于)：水、盐水、磷酸缓冲液或其它水性溶剂；DMSO(二甲基亚砜)、甘油和乙醇或其它有机溶剂；微球、脂质体、微乳液或高分子表面活性剂；胶体型载药***或高分子载药***；或防腐剂、抗氧剂、矫味剂、芳香剂、助溶剂、乳化剂、pH缓冲物质，黏合剂、填充剂、润滑剂或其它药物赋形剂。
在另一优选例中，所述的组合物可制备的药物剂型包括(但不限于)：固体剂型，包括(但不限于)粉剂、散剂、片剂、丸剂、胶囊剂、缓释剂、控速释剂；液体剂型，包括(但不限于)注射剂、输液剂、混悬剂，或其它液体剂型；气体剂型；或半固体剂型。
在另一优选例中，所述的组合物可添加水性溶剂或有机溶剂配制成为科研用试剂；可添加基础培养基或无血清培养基制备成诱导肿瘤细胞直接重编程为非致瘤性细胞的培养基，组合物中各成分存在于含5-20％小牛血清、1％青链霉素混合液(100x)的基础细胞培养基或含有各种细胞因子或生长因子的无血清培养基中，但组合物中不包括细胞分化基本培养基。
在本发明的另一方面，提供所述的组合物的用途，用于研发或制备***的药物(或药物配方)；或用于制备诱导人肿瘤细胞直接重编程(转分化)为非致瘤性细胞并伴随肿瘤细胞凋亡的培养基或试剂。
在本发明的另一方面，提供一种诱导肿瘤细胞直接重编程为非致瘤性细胞并伴随肿瘤细胞凋亡的方法，其特征在于，所述方法包括：应用权利要求1-6任一所述的组合物诱导调控人肿瘤细胞直接转分化为非致瘤性细胞，并伴随肿瘤细胞凋亡。
在另一优选例中，提供制备诱导人肿瘤细胞转分化并伴随凋亡的培养基或试剂方法及其实验步骤，包括：
(1)浓缩液试剂配制：根据权利要求1-6任一所述的组合物，将各成分溶解于有机溶剂或水性溶剂中配制成浓缩液试剂；较佳地，所述的有机溶剂包括二甲基亚砜；较佳地，所述的水性溶剂包括：水，生理盐水，磷酸盐缓冲液；
(2)培养基获得：将步骤(1)中的浓缩液试剂分别稀释入含5-20％小牛血清、1％青链霉素混合液(100x)的基础细胞培养基或含有各种细胞因子或生长因子的无血清培养基中(使得各组分的浓度符合权利要求1-6任一所述的组合物中所限定的浓度)，获得诱导肿瘤细胞转分化伴随凋亡的培养基；其中，该培养基各组分的百分含量还可上下浮动50％；较佳地上下浮动30％；更佳地上下浮动20％，如10％，5％；
(3)诱导肿瘤细胞转分化伴随凋亡：将肿瘤细胞在上述步骤(2)配制的诱导“肿瘤细胞转分化伴随凋亡培养基”中混悬，铺板作为处理组；将与处理组培养基溶解小分子组合 物的溶剂(如DMSO或其它溶剂)等量地添加入同处理组所用含10％小牛血清、1％青链霉素混合液(100x)的基础细胞培养基(或含有各种细胞因子或生长因子的无血清培养基)中，获得“对照培养基”(百分数均以v/v计)；然后将与处理组等数量的肿瘤细胞加入“对照培养基”中混悬，铺板，作为对照组；37℃培养，每2-4天换液一次；3-7天传代一次。
(4)诱导肿瘤细胞转分化伴随凋亡的传代培养：弃原培养液，PBS洗涤一次，加入细胞消化液消化细胞，37℃，1-5分钟，终止细胞消化，离心，弃上清，将细胞沉淀重悬，按1:1-1:3传代铺板。按实验步骤第1和2项方法培养，每2-4天换液一次。所用消化液包括胰酶，EDTA,Acutase,TrypleE等。3-7天传代一次。
(5)诱导肿瘤细胞转分化伴随凋亡获得正常或非致瘤性细胞：经上述实验步骤(3)、(4)转分化伴随凋亡培养和传代培养肿瘤细胞1-4周，PBS洗涤去除凋亡细胞，即可获得转分化后的非致瘤性细胞。
在本发明的另一方面，提供一种用于诱导人肿瘤细胞直接重编程为非致瘤性细胞并伴随肿瘤细胞凋亡的药盒/试剂盒，所述的药盒/试剂盒中包括：前面任一所述的组合物；或基于该组合物开发制备的***的药物或药物配方；或基于该组合物制备的科研用试剂或培养基。
如前面任一方面，所述的肿瘤或肿瘤细胞包括但不限于：肝癌、肺癌、胃癌、结直肠癌、胰腺癌、乳腺癌、卵巢癌、***癌、骨肉瘤、淋巴瘤、白血病、鼻咽癌、食管癌、***、口腔癌、唾液腺肿瘤、鼻腔与鼻旁窦恶性肿瘤、喉癌、耳部肿瘤、眼部肿瘤、甲状腺肿瘤、纵隔肿瘤、胸壁、胸膜肿瘤、小肠肿瘤、胆道肿瘤、胰腺与壶腹周围肿瘤、肠系膜与腹膜后肿瘤、肾脏肿瘤、肾上腺肿瘤、***、睾丸肿瘤、***癌、子宫内膜癌、卵巢恶性肿瘤、恶性滋养细胞肿瘤、外阴癌与***癌、恶性淋巴瘤、多发性骨髓瘤、软组织肿瘤、骨肿瘤、皮肤及附件肿瘤、恶性黑色素瘤或神经***肿瘤及其它血液***肿瘤和实质性肿瘤或其细胞；较佳地为肝癌或肝癌细胞。
本发明的其它方面由于本文的公开内容，对本领域的技术人员而言是显而易见的。
附图说明
图1、小分子(培养基6)诱导肝癌细胞SMMC-7721转分化为非致瘤性细胞，并伴随凋亡。
A图、肝癌细胞SMMC-7721被培养基6培养10天诱导转分化为非致瘤性肝细胞样细胞，形态完全改变，显示已被转分化；
B图、肝癌细胞SMMC-7721被培养基6诱导转分化伴随凋亡培养1-14天的凋亡统计。
图中蓝色柱代表早期(Early)凋亡，红色柱代表晚期(Late)凋亡；T、T1W、T2W分别代表直接处理、处理1、2周的肝癌细胞SMMC-7721凋亡情况。对照组和处理组的 凋亡值具统计学差异(p<0.05)。
图2、肝癌细胞HepG2被培养基4、1诱导转分化并伴随凋亡。
A、肝癌细胞HepG2被培养基4，培养8天诱导转分化为非致瘤性肝细胞样细胞，形态完全改变，显示已被转分化；
B、肝癌细胞HepG2被培养基1诱导转分化伴随凋亡。
图中蓝色柱代表早期凋亡，红色柱代表晚期凋亡；T、T1W、T3W分别代表直接处理、处理1、3周的凋亡检测结果。对照组和处理组的凋亡值具统计学差异(p<0.05)。
图3、耐5-Fu肝癌细胞7402/5-Fu被培养基5、2诱导转分化伴随凋亡。
A、右图处理组肝癌细胞7402/5-Fu被培养基5处理2周诱导转分化后形态完全改变，显示已被转分化；
B、肝癌细胞7402/5-Fu被培养基2诱导转分化伴随凋亡。图中蓝色柱代表早期凋亡，红色柱代表晚期凋亡；T2W、T3W、T4W分别代表处理2、3、4周的凋亡检测结果。对照组和处理组的凋亡值具统计学差异(p<0.005)。
图4、肝癌细胞SMMC-7721(培养基6)，HepG2(培养基4)，7402/5-Fu(培养基5)被诱导转分化为非致瘤性肝细胞样细胞并具正常肝细胞功能。PAS：糖原染色，Oil-red：油红染色，反映脂肪摄取功能。
图5、肝癌细胞SMMC-7721(培养基10)、HepG2(培养基11)、7402/5-Fu(培养基12)被诱导转分化为非致瘤性肝细胞样细胞并具正常肝细胞白蛋白分泌(ALB)，尿素生成(Urea)，CYP1A2诱导和CYP3A4诱导功能。
A、肝癌细胞SMMC-7721被培养基10诱导转分化获得的非致瘤性肝细胞样细胞具有正常肝细胞功能；
B、肝癌细胞HepG2被培养基11诱导转分化获得的非致瘤性肝细胞样细胞具有正常肝细胞功能；
C、耐5-Fu肝癌细胞7402/5-Fu被培养基12诱导转分化获得的非致瘤性肝细胞样细胞具有正常肝细胞功能；
T1W、T2W、T3W分别代表处理1、2、3周。Rif：利福平；Ome：奥美拉措。
图6、肝癌细胞SMMC-7721(培养基6)，HepG2(培养基4)和7402/5-Fu(培养基5)被诱导转分化获得的非致瘤性肝细胞样细胞体内体外均不再具致瘤性。
A、肝癌细胞SMMC-7721，HepG2和7402/5-Fu分别被培养基6、4、5诱导转分化获得的非致瘤性肝细胞样细胞体外生长不形成克隆，体外失去致瘤性；
B、肝癌细胞SMMC-7721被诱导转分化获得的非致瘤性肝细胞样细胞体内不生成肿瘤(处理4周)，体内失去致瘤性(培养基6)。
C、肝癌7402/5-Fu被诱导转分化获得的非致瘤性肝细胞样细胞体内不生成肿瘤(处理4周)，体内失去致瘤性(培养基5)；C图上图裸鼠后腿右侧为处理组细胞不形成肿瘤，失去致瘤性；后腿左侧对照组细胞形成肿瘤；C图下图为对照组形成的肿瘤解剖形状 外观。
图7、小分子组合体内应用于病人肝癌组织PDX动物模型试验(转分化伴随凋亡组合物8)。结果显示：处理组瘤体组织坏死，组织结构已被破坏或丧失(红色染色)；对照组瘤体组织、细胞结构无变化(***染色)(PDX-80872)。图中4、7、8等数字表示小鼠耳钉代码。
图8、正常人成纤维细胞和肝细胞被(转分化伴随凋亡培养基8、3)处理培养3周后，形态无改变，显示其不受影响。
图9、鼻咽癌细胞HNE和肺癌细胞H460分别被培养基9、7诱导转分化伴随凋亡。由图9可见，鼻咽癌细胞HNE(培养基9处理)处理组癌细胞转分化后形态完全改变，显示其已转分化；处理组肺癌细胞H460被(培养基9、7)分别处理，其中肺癌细胞H460被(培养基7)几乎全部诱导凋亡(图中绿色柱代表早期凋亡，红色柱代表晚期凋亡；T10D、T20D分别代表处理10天、处理20天)；而对照组肺癌细胞H460几乎无凋亡。
图10、胃癌细胞SGC-7901和MKN28分别被诱导转分化(培养基12、13处理2周)。胃癌细胞SGC-7901(培养基12)和MKN28(培养基13)分别处理2周，处理组的癌细胞转分化后形态完全转变，显示其已被转分化；
图11、胰腺癌细胞SW1990被诱导转分化(培养基12处理2周)。胰腺癌细胞SW1990处理组的癌细胞转分化后，形态完全改变，显示其已被转分化；
图12、乳腺癌细胞SKBr3被诱导转分化(培养基13处理2周)。乳腺癌细胞SKBr3处理组的癌细胞转分化后，形态完全转变，显示其已被转分化；
图13、白血病细胞U937、B细胞淋巴瘤SUDHL-4被诱导转分化伴随凋亡(培养基10、11处理10天)。白血病细胞U937(培养基10)、B细胞淋巴瘤SUDHL-4(培养基11)处理组的癌细胞大多被诱导凋亡，细胞形态不再完整。
图14、乳腺癌细胞SKBr3和胃癌细胞MKN28被诱导转分化(培养基13、14)。乳腺癌细胞SKBr3和胃癌细胞MKN28分别被培养基13、14处理2周，图14右上下图显示处理组(T)的癌细胞形态完全改变，显示其已被转分化。
图15、小分子组合诱导肠癌HCT116细胞转分化后失去致瘤性。肠癌细胞HCT116被培养基9处理2周，右图显示肠癌细胞HCT116处理组的癌细胞形态完全改变，且不再形成克隆，显示其已转分化，失去致瘤性。
图16、小分子诱导***癌PC-3细胞转分化后失去致瘤性。***癌PC-3细胞被培养基5处理2周，图16右图显示处理组的胰腺癌PC-3细胞被诱导转分化后不再形成克隆，失去体外致瘤性。
图17、小分子诱导卵巢癌细胞SKOV3和A2780细胞转分化后失去致瘤性。右侧上下图分别显示处理组的卵巢癌细胞SKOV3和A2780分别被培养基7、8诱导转分化后不再形成克隆，失去致瘤性。
图18、小分子诱导胃癌、乳腺癌细胞转分化后失去致瘤性。右侧上下图分别显示 处理组的胃癌细胞MKN28，乳腺癌细胞SKbr3，分别被培养基10、11分别处理2周，诱导转分化后不再形成克隆，失去致瘤性。
图19、小分子诱导神经胶质瘤细胞转分化后形态完全改变。右上下图处理组神经胶质瘤细胞T98G、U87MG被培养基4、5分别处理2周，分别诱导转分化后形态完全改变，显示其已转分化。
图20、小分子组合体内应用于肝癌PDX动物模型实验。左图处理组的肝癌组织及细胞，经转分化伴随凋亡小分子组合物21配制的注射用试剂21处理3周后，大面积坏死，癌组织结构已被破坏或丧失。右图处理组显示，经小分子试剂处理3周后，残留组织及细胞表达人肝细胞特有标志物HNF4a，提示已发生转分化。
图21、小分子诱导肺癌细胞转分化后体内外失去致瘤性。A为培养基处理2周结果，下图处理组显示，肺癌细胞A549、H1299和H460分别被培养基14、13、9诱导转分化后不再形成克隆，体外失去致瘤性；B中，裸鼠右侧后腿(蓝色箭头所指)为处理组肺癌细胞转分化后注射入裸鼠体内4周，处理组显示转分化后的细胞不再形成肿瘤，表明肺癌细胞A549(培养基14处理)被诱导转分化后，体内也失去致瘤性。红色箭头所指为未处理肺癌细胞形成的肿瘤。
图22、小分子诱导肺癌细胞H1299转分化后，体外失去致瘤性。右图显示处理组的肺癌细胞H1299(培养基15处理2周)转分化后不再形成克隆，失去致瘤性。
图23、卵巢癌细胞A2780、SKOV3被分别诱导转分化后体外失去致瘤性。右上下图分别显示处理组的卵巢癌细胞A2780(培养基16)、SKOV3(培养基17)处理2周，转分化后不再形成克隆，失去致瘤性。
图24、***癌细胞PC9被培养基18处理2周诱导转分化。右图处理组的***癌细胞PC9被诱导转分化后，细胞形态完全改变，显示其已转分化。
图25、胃癌SGC-7901细胞被培养基19、20分别诱导转分化。A图处理组显示胃癌SGC-7901细胞(培养基19处理2周)转分化后不再形成克隆，体外失去致瘤性。B图胃癌SGC-7901细胞被诱导凋亡(培养基20处理2周)统计结果。图中绿色柱代表早期凋亡(Early)，红色柱代表晚期凋亡(Late)；T1W、T2W分别代表处理1、2周的凋亡统计；对照组有很少部分早、晚期自然凋亡。对照组和处理组的凋亡值具统计学差异(p<0.005)。
图26、胰腺癌SW1990细胞被培养基16处理2周诱导转分化后失去致瘤性，右图处理组的胰腺癌SW1990细胞，转分化后不再形成克隆，体外失去致瘤性。
具体实施方式
本发明人经过深入的研究，揭示了一种小分子组合物诱导人肿瘤细胞直接重编程(转分化)为非致瘤性细胞并伴随肿瘤细胞凋亡的方法及其小分子组合物；该小分子组合物可添加药物载体或赋型剂开发制备成临床***的药物或药物配方；可添加水性 溶剂或有机溶剂、基础培养基或无血清培养基制备成科研用试剂或培养基。该小分子组合物诱导人肿瘤细胞直接重编程(转分化)方法首先应用于肝癌细胞，将肝癌细胞直接转分化为非致瘤性肝细胞样细胞并伴随肝癌细胞不同程度凋亡；经转分化获得的肝细胞样细胞具有正常肝细胞功能；在已实验测试范围内对正常肝细胞和成纤维细胞无损伤。在此基础上，该小分子组合物还将肺癌、胃癌、胰腺癌、乳腺癌、白血病、淋巴癌、神经胶质瘤等为代表的各种肿瘤或肿瘤细胞转分化为非致瘤性细胞，并伴随不同程度的肿瘤细胞凋亡。
基本机理
化学诱导细胞直接重编程(转分化)是指不改变基因序列的情况下，通过调控细胞信号通路及表观遗传的变化来改变细胞命运的过程。
随着干细胞科学发展，细胞重编程在不断创新，已从外源导入转录因子实现重编程；到现在使用调控细胞信号通路和表观遗传的小分子组合也可实现“化学诱导多能细胞(iPSC)重编程”(Hongkui Deng等，Pluripotent Stem Cells Induced from Mouse Somatic Cells by Small-Molecule Compounds.Science.341,651-4,2013)和“化学诱导细胞直接重编程”(Li X,Zuo X,Jing J,Ma Y,Wang J,Liu D,Zhu J,Du X,Xiong L,Du Y,Xu J,Xiao X,Chai Z,Zhao,Y,Deng,H.Small-Molecule-Driven Direct Reprogramming of Mouse Fibroblasts into Functional Neurons.Cell Stem Cell.17(2):195-203,2015；Hu W等Direct Conversion of Normal and Alzheimer’s Disease Human Fibroblasts into Neuronal Cells by Small Molecules.Cell Stem Cell.17(2):204-212,2015)；重编程的起始和目的细胞类型也从分化细胞重编程为多能干细胞，扩展到直接重编程为另一种分化细胞；甚至可从异常细胞(粘液瘤细胞)外源导入转录因子重编程为多能样细胞，但仍保有其致瘤性(Zhang X,Cruz FD,Terry M,Remotti F and Matushansky I.Terminal differentiation and loss of tumorigenicity of humancancers via pluripotency-based reprogramming.Oncogene,2,2249–2260,2013)；因此，仅使用小分子将肿瘤细胞直接重编程为非致瘤性细胞已成为可能。
表1 多种起始细胞重编程为同一个目的细胞实例
Figure PCTCN2016107910-appb-000001
从已报道通过细胞重编程将多种类型的正常分化细胞重编程为同一种类型的细胞实例(表1)可以看出，起始细胞的类型不是关键，筛选能够构建并维持目的细胞特有的基因表达谱或生物学行为，以及能够突破起始细胞重编程过程中的各种“能障”的诱导因 子组合，才是细胞重编程关键。
因此，只要找到能够突破各种“能障”和构建并维持目的细胞特有基因表达谱的诱导因子或化合物，不仅不同胚层的各种分化细胞，甚至包括肿瘤细胞，就都可能通过细胞重编程完成细胞命运的转变。因此，按所述思路筛选小分子组合，诱导调控肿瘤细胞转分化为相应的非致瘤性细胞(目的细胞)，并能够克服肿瘤细胞异质性，理论上是可行的。
本发明首先选择异质性最强的肝癌为突破点。筛选出能够构建并维持正常(非致瘤性)肝细胞(目的细胞)特有基因表达谱，同时能够突破肝癌细胞向正常肝细胞转分化过程中的各种“能障”的小分子组合，构建了小分子诱导肝癌细胞转分化为非致瘤肝细胞，并伴随不同程度的肝癌细胞凋亡的方法平台；该创新方法具有以下优点：①小分子性质稳定，作用的时间、剂量及组合方式易于控制，作用效果稳定可靠；②转分化后的肝细胞样细胞不但具有正常成熟的人肝细胞功能，而且体内外不再具致瘤性；③该方法能强有力地转分化肝癌细胞且对正常肝细胞及成纤维细胞没有损伤；也不需杀细胞试剂辅助杀灭肿瘤细胞，因此避免了对正常组织细胞的无区别杀伤副作用；④不需要导入外源基因或改变细胞基因结构，避免了外源基因导入或基因改变引起新的致癌风险，所以安全可靠；⑤该方法采取化学诱导直接重编程，不需要重编程为诱导多能干细胞(iPSC)，避免了干细胞的致癌风险。
该方法对致病原因及发病机理复杂，异质性强且缺乏有效治疗手段的人体肝癌的控制或有效治疗具有重要意义。不仅如此，进一步研究结果显示，该小分子组合物还对诱导鼻咽癌、肺癌、胃癌、结直肠癌、胰腺癌、乳腺癌、卵巢癌、***癌、骨肉瘤、淋巴瘤、白血病以及其他血液***肿瘤和实质性肿瘤细胞直接重编程(转分化)为非致瘤性细胞，并伴随肿瘤细胞不同程度凋亡，具有相同或类似效果。因此，上述系列实验结果验证了本发明人设计用小分子组合多靶点诱导肿瘤细胞直接重编程(转分化)为非致瘤性细胞，而不能转分化的肿瘤细胞则伴随凋亡。这一设计思路和方案是成功有效的。
药物组合物及其应用
本发明人经过广泛的研究，首次提出同时不同程度地抑制GSK3β和TGFβ信号通路即可诱导肿瘤细胞转分化并伴随肿瘤细胞凋亡。根据肿瘤细胞异质性的个体差异或肿瘤细胞株之间的差异，被诱导转分化的肿瘤细胞与被诱导凋亡的肿瘤细胞数目比例有不同变化；有的偏重于转分化，有的偏重于凋亡；而同时抑制GSK3β和TGFβ信号通路偏重于凋亡，转分化不够彻底。
而在抑制GSK3β和TGFβ信号通路的同时结合维甲酸(RA)类化合物的协调作用，则能够增强小分子组合物诱导肿瘤细胞转分化为非致瘤性细胞，同时伴随肿瘤细胞不同程度的凋亡。若再联合BMP信号通路的下调，和/或联合BrdU(或/和EdU)，则 可进一步增强或促进某些恶性更强的肿瘤细胞转分化并伴随肿瘤细胞凋亡。尽管因肿瘤的异质性不同而被小分子组合转分化与凋亡的比例有所不同。但不影响该小分子组合诱导肿瘤细胞转分化为非致瘤性细胞并伴随肿瘤细胞凋亡的最终效果。因此，该小分子组合物有潜力开发为抗肿瘤治疗的新药物或药物配方；并可直接制备为化学诱导肿瘤细胞直接重编程为非致瘤性细胞并伴随肿瘤细胞凋亡的科研用培养基或试剂。
应理解，除了本发明实施例中所列举的具体GSK3β抑制剂、TGFβ抑制剂、BMP抑制剂以外的其它可抑制GSK3β信号通路、TGFβ信号通路、BMP信号通路的，诱导调控相同靶点的，或具相同功能的同种类抑制剂也可实现同样的技术效果，也应被包含在本发明中。
同样，除了本发明实施例中所列举的具体维甲酸(RA)诱导分化剂以外的其它具相同功能的，或诱导调控相同靶点的维甲酸类化合物也可实现同样的技术效果；也应被包含在本发明中。
如本文所用，术语“含有”或“包括”包括了“包含”、“基本上由……构成”、和“由……构成”。
如本文所用，术语“基本上由……构成”指在组合物中，除了含有必要成分或必要组份之外，还可含有少量的且不影响有效成分的次要成分和/或杂质。例如，可以含有甜味剂以改善口味、抗氧化剂以防止氧化，以及其他本领域常用的药物添加剂、载体、赋形剂。
如本文所用，术语“药学上可接受的”的成分是适用于人和/或动物而无过度不良副反应(如毒性、刺激和***反应)的，即有合理的效益/风险比的物质；如本领域常用的药物载体或赋形剂。
如本文所用，术语“有效量”是指可对人和/或动物产生功能或活性的且可被人和/或动物所接受的量。
如本文所用，术语“药学上可接受的载体或赋形剂”，其中载体指能改变药物进入人体的方式和在体内的分布、控制药物的释放速度并将药物输送到靶向器官的体系；药物载体本身并不是必要的活性成分，且施用后没有过分的毒性。合适的载体是本领域普通技术人员所熟知的，包括但不限于：水、盐水、磷酸缓冲液以及其它水性溶剂；DMSO(二甲基亚砜)、甘油和乙醇以及其它有机溶剂；微球、脂质体、微乳液、高分子表面活性剂；胶体型载药***、新型高分子载药***、新型药物载体以及其他药学上的载体；其中赋形剂指在药物制剂中除主药以外的附加物，也可称为辅料。如片剂中的黏合剂、填充剂、崩解剂、润滑剂；中药丸剂中的酒、醋、药汁等；半固体制剂软膏剂、霜剂中的基质部分；液体制剂中的防腐剂、抗氧剂、矫味剂、芳香剂、助溶剂、乳化剂、增溶剂、渗透压调节剂、着色剂等均可称为赋形剂。
对赋形剂的一般要求是性质稳定，与主药无配伍禁忌，不产生副作用，不影响疗效，在常温下不易变形、干裂、霉变、虫蛀、对人体无害、无生理作用，不与主药产生化学 或物理作用，不影响主药的含量测定等。在Remington’s Pharmaceutical Sciences(Mack Pub.Co.，N.J.1991)中可找到关于药学上可接受的载体或赋形剂的充分讨论。其载体或赋形剂包括但不限于：水、盐水、磷酸缓冲液等水溶液；DMSO(二甲基亚砜)、甘油和乙醇等有机溶剂；微球、脂质体、微乳液、高分子表面活性剂；胶体型载药***、新型高分子载药***、新型药物载体以及其他药学上的载体；液体制剂中的防腐剂、抗氧剂、矫味剂、芳香剂、助溶剂、乳化剂、pH缓冲物质，片剂中的黏合剂、填充剂、润滑剂以及其它药物赋形剂等。
如本文所用，术语“组合物可制备的药物剂型”中的药物剂型指：为适应治疗或预防的需要而制备的药物应用形式，称为药物剂型；本发明任一组合物可制备的药物剂型包括但不限于：粉剂、散剂、片剂、丸剂、胶囊剂、缓释剂、控速释剂及其它固体剂型；注射剂、输液剂、混悬剂及其它液体剂型，以及气体剂型、半固体剂型等其它剂型。
如本文所用，“重量份”或“重量份数”可互换使用，所述的重量份可以是任何一个固定的以微克、毫克、克数或千克数表示重量(如1ug、1mg、1g、2g、5g、或kg等)。例如，一个由1重量份组分a和9重量份组分b构成的组合物，可以是1克组分a+9克组分b，也可以是10克组分a+90克组分b等构成的组合物。在所述组合物，某一组分的百分比含量＝(该组分的重量份数/所有组分的重量份数之和)×100％。因此，由1重量份组分a和9重量份组分b构成的组合物中，组分a的含量为10％，组分b为90％。
此外，在溶液状态时，上述“重量份”也可以换算成为“摩尔数”；“重量份比”也可以换算成为“摩尔浓度比”。所述重量份比的重量单位可以是：千克(kg)、毫克(mg)、微克(ug)等任一重量单位；摩尔(浓度)比的摩尔单位可以是：摩(M)、毫摩(mM)、微摩(uM)等任一摩尔浓度单位；
在一个优选例中：所述的组合物中，GSK3β抑制剂(或GSK3β抑制剂CHIR99021)、TGFβ抑制剂(或TGFβ抑制剂SB431542或/和A83-01)以重量份比：(0.046-4.65)﹕(0.038-7.68)；较佳地，(0.232-2.325)﹕(0.192-3.84)存在；或以摩尔比：(0.1-10)﹕(0.1-20)；较佳地，(0.5-5)﹕(0.5-10)存在。
在另一优选例中：所述的组合物：GSK3β抑制剂(或GSK3β抑制剂CHIR99021)，TGFβ抑制剂(或TGFβ抑制剂SB431542或/和A83-01)，和维甲酸类化合物(或维甲酸)以重量份比：(0.046-4.65)﹕(0.038-7.68)﹕(0.03-6.0)；较佳地，(0.232-2.325)﹕(0.192-3.84)﹕(0.15-3)存在；或以摩尔比：(0.1-10)﹕(0.1-20)﹕(0.1-20)；较佳地，(0.5-5)﹕(0.5-10)﹕(0.5-10)存在。
在另一优选例中，所述的组合物中，GSK3β抑制剂(如GSK3β抑制剂CHIR99021)，TGFβ抑制剂(如TGFβ抑制剂SB431542或/和A83-01)，维甲酸类化合物(如维甲酸)，BMP抑制剂(如BMP抑制剂LDN-193189)和BrdU以重量份比：(0.046-4.65)﹕ (0.038-7.68)﹕(0.03-6.0)﹕(0.02-4.65)﹕(0.15-30)；较佳地，(0.232-2.325)﹕(0.192-3.84)﹕(0.15-3)﹕(0.203-2.03)﹕(1.5-15)存在；或溶液状态下以摩尔比：(0.1-10)﹕(0.1-20)﹕(0.1-20)﹕(0.05-10)﹕(0.5-100)；较佳地，(0.5-5)﹕(0.5-10)﹕(0.5-10)﹕(0.5-5)﹕(5-50)存在。
在另一优选例中，所述的组合物中，GSK3β抑制剂(如GSK3β抑制剂CHIR99021)，TGFβ抑制剂(如TGFβ抑制剂SB431542或/和A83-01)，维甲酸类化合物(如维甲酸)，BMP抑制剂(如BMP抑制剂LDN-193189)和EdU以重量份比：(0.046-4.65)﹕(0.038-7.68)﹕(0.03-6.0)﹕(0.02-4.65)﹕(0.125-25)；较佳地，(0.232-2.325)﹕(0.192-3.84)﹕(0.15-3)﹕(0.203-2.03)﹕(1.25-12.5)存在；或溶液状态下以摩尔比：(0.1-10)﹕(0.1-20)﹕(0.1-20)﹕(0.05-10)﹕(0.5-100)；较佳地，(0.5-5)﹕(0.5-10)﹕(0.5-10)﹕(0.5-5)﹕(5-50)存在。
作为本发明的优选方式，所述的组合物中，包括的成分及其重量份比或摩尔浓度比如表2或表3(溶液状态组合物)所示。
表2
Figure PCTCN2016107910-appb-000002
表3
Figure PCTCN2016107910-appb-000003
表1和表2的配方范围可以作为参考性指导。但是应理解，当用于开发制备药物组合物时，所用的组合物的有效剂量可随施用的模式和待***的种类以及疾病的 严重程度而变化。且，体内使用时，通常使用“重量/公斤(体重)”作为剂量单位；所述的小分子组合物应用于大动物和肿瘤病人时，按小动物使用剂量通过相应的专业换算公式，换算出的大动物或人的有效使用剂量(包括固态或溶液态的剂量换算)，也属于本发明的保护范围。
如本发明所用，所述的“GSK3β抑制剂”是指能够抑制细胞中GSK3β信号通路的抑制剂的总称，包括但不限于：CHIR-99021，BIO、IM-12、TWS119等为代表的，具相同功能，或诱导相同靶点的同一类GSK3β信号通路抑制剂：
CHIR-99021(CT99021)，其是GSK-3α和β抑制剂，IC50分别为10nM和6.7nM，比对CDC2，ERK2及其他激酶的抑制性强500倍；
CHIR-99021(CT99021)HCl，其是CHIR-99021的盐酸盐，是一种GSK-3α/β抑制剂，在无细胞试验中IC50为10nM/6.7nM，可用于区分GSK-3和其最接近的同源物Cdc2与ERK2；
BIO，其是一种特异性的GSK-3抑制剂，无细胞试验中作用于GSK-3α/β的IC50为5nM；
IM-12，其是一种选择性的GSK-3β抑制剂，其IC50为53nM，增强了Wnt信号通路；
TWS119，其是一种GSK-3β抑制剂，在无细胞试验中IC50为30nM；
1-Azakenpaullone，其是一种高度选择性GSK-3β抑制剂，IC50为18nM；
CHIR-98014，其是一种有效的GSK-3α/β抑制剂，无细胞试验中IC50为0.65nM/0.58nM；
Tideglusib，其是一种不可逆的，非ATP竞争性的GSK-3β抑制剂，无细胞试验中IC50为60nM；
AR-A014418，其是一种ATP竞争性和选择性的GSK3β抑制剂，无细胞试验中IC50和Ki为104nM和38nM；
LY2090314，其是一种有效的GSK-3抑制剂，作用于GSK-3α/β，IC50为1.5nM/0.9nM；
SB216763，其是一种有效的，选择性GSK-3α/β抑制剂，IC50为34.3nM；
AZD1080，其是一种口服生物有效的，选择性的，可透过大脑的GSK3抑制剂，抑制人类GSK3α和GSK3β，Ki分别为6.9nM和31nM，比作用于CDK2，CDK5，CDK1和Erk2选择性高14倍以上。
作为本发明的优选方式，所述的GSK3β抑制剂是CHIR-99021，其别名为CT99021；其分子结构式如以下式(I)所示：
Figure PCTCN2016107910-appb-000004
如本发明所用，所述的“TGFβ抑制剂”是指能够抑制细胞中TGFβ信号通路的抑制剂的总称，包括但不限于：SB431542、A83-01、SB525334、LY2109761、RepSox等为代表的，具相同功能，或诱导相同靶点的同一类TGFβ信号通路抑制剂：
SB-431542，其是有效的，选择性的ALK5抑制剂，IC50为94nM，比对p38、MAPK和其他激酶的抑制性强100倍；
A83-01，其是ALK5，ALK4和ALK7的抑制剂，IC50分别为12，45和7.5nM；
SB525334，其是一种有效的，选择性TGFβreceptor I(ALK5)抑制剂，无细胞试验中IC50为14.3nM，作用于ALK4比作用于ALK5效果低4倍，对ALK2，3，和6没有活性；
LY2109761，其是一种新型的，选择性TGF-βreceptor type I/II(TβRI/II)双重抑制剂，无细胞试验中Ki分别为38nM和300nM；
RepSox，其是一种有效的，选择性TGFβR-1/ALK5抑制剂，作用于ATP与ALK5结合以及ALK5自磷酸化，无细胞试验中IC50分别为23nM和4nM。
SD-208，其是一种选择性TGF-βRI(ALK5)抑制剂，with IC50为48nM，选择性比TGF-βRII高100多倍；
GW788388，其是一种有效的，选择性ALK5抑制剂，无细胞试验中IC50为18nM，也抑制TGF-βII型受体和activin II型受体活性，但不抑制BMP II型受体；
SB505124，其是一种选择性TGFβR抑制剂，作用于ALK4和ALK5，无细胞试验中IC50分别为129nM和47nM，也抑制ALK7，但不抑制ALK1，2，3或6；
EW-7197，其是一种高效的，选择性的，口服生物有效的TGF-βreceptor ALK4/ALK5抑制剂，IC50分别为13nM和11nM。
作为本发明的优选方式，所述的TGFβ抑制剂是SB 431542(或称为SB-431542)；其分子结构式如以下式(II)所示：
Figure PCTCN2016107910-appb-000005
作为本发明的优选方式，所述的TGFβ抑制剂是A83-01(或称为A8301)；其分子结构式如以下式(III)所示：
Figure PCTCN2016107910-appb-000006
如本发明所用，所述的维甲酸类(retinoids)化合物，包括但不限于：维甲酸(Retinoic acid，RA)，别名：全反式维甲酸(all trans retinoic acid，ATRA)；13-顺式维甲酸(13-cis retinoic acid，13-CRA)、9-顺式维甲酸(9-cis-retinoic acid，9-CRA)等为代表的，具 相同功能，或诱导相同靶点的同一类维甲酸类诱导分化剂或化合物，或其组合。
维甲酸类(retinoids)化合物具有调节细胞增殖、分化和细胞生理凋亡(apo ptosi s)的功能，因其能够激活相应的维甲酸核受体(retinoie acid receptor，RAR)和维甲酸X核受体(retinoid x receptor，RXR)蛋白，通过与维甲酸应答因子(retinoic acid respo nse elements，RARE)特异性结合，从而调控特定核基因的转录活性，产生生物学效应。维甲酸类化合物及其异构体衍生物中许多都具有相同或相类似的功能，因此成为一类重要的诱导分化剂或化合物。
维甲酸类(retinoids)化合物是一组维生素A(视黄醇)的氧化代谢产物或衍生物以及与维生素A具有类似结构的人工合成物,包括天然和合成两大类。主要包括：维甲酸(Retinoic acid，RA)(别名：维A酸、全反式维甲酸)、13-顺式维甲酸(13-cis retinoic acid，13-CRA)、阿维A酯、9-顺式维甲酸(9-cis-retinoic acid，9-CRA)、UAB7、UAB8、异维甲酸(Isotretinoin)、维胺酯(Fenretinide)、依曲替酸(Acitretin)、依曲替酯(Etretinate)、他扎罗汀(Tazarotene)、阿达帕林(Adapalene)、TTNPB、3-甲基-TTN PB、AM80、AM580、CD437、Targretin、LGD1069及其他具相同功能的维甲酸类化合物。其中RA或ATRA、TTNPB，AM80和AM580是RAR的特异性激动剂；LGD 1069和SR 11237是RXR的特异性激动剂；9-CRA和3-甲基-TTNPB则可激动这两类受体蛋白，属于泛激动剂。
作为本发明的优选方式，所述的维甲酸(Retinoic acid，RA)，别名：全反式维甲酸(all trans retinoic acid，ATRA)、维A酸、维生素A酸、维生素甲酸、视黄酸、全反式维A酸、维A甲酸，其分子结构式如以下式(IV)所示：
Figure PCTCN2016107910-appb-000007
如本发明所用，所述的“BMP抑制剂”是指能够抑制细胞中BMP信号通路的抑制剂的总称，包括但不限于：LDN-193189、LDN193189HCl、K02288、DMH1等为代表的，具相同功能，或诱导相同靶点的同一类BMP信号通路抑制剂：
LDN-193189，是一种选择性的BMP信号通路抑制剂，抑制BMP I型受体ALK2和ALK3的转录活性，在C2C12细胞中IC50分别为5nM和30nM，作用于BMP比作用于TGF-β选择性高200倍。Cas：1062368-24-4；
LDN193189HCl，是LDN193189的盐酸盐，是一种选择性BMP信号抑制剂，它能抑制BMP type I receptors ALK2和ALK3的转录活性，在C2C12细胞中IC50分别为5nM和30nM，并且对于BMP的选择性是对TGF-β选择性的200倍。
K02288，是一种高度选择性I型BMP受体抑制剂，针对ALK2，ALK1和ALK6的IC50分别为1.1，1.8，6.4nm，显示对其他ALKS(3，4，5)和ActRIIa有轻微抑制作用。
DMH1，是选择性BMP receptor抑制剂，抑制ALK2，IC50为107.9nM,对AMPK， ALK5，KDR(VEGFR-2)和PDGFR没有抑制效果。
作为本发明的优选方式，所述的BMP抑制剂是LDN-193189(或称为LD-N193189)；其分子结构式如以下式(Ⅴ)所示：
Figure PCTCN2016107910-appb-000008
如本发明所用，所述的BrdU，中文全名5-溴脱氧尿嘧啶核苷，别名：5-溴-2’-脱氧尿苷；5-溴-2-脱氧脲苷；5-溴-1-(2-脱氧-β-D-呋喃核糖)尿嘧啶；5-溴脱氧尿苷；溴化去氧尿苷；英文名称：5-Bromo-2"-Deoxyuridine；简称：BrdU或5-BrdU。别名：5-Bromo-2-deoxyUridine；Br-dU；BUdR；5-Bromodeoxyuridine；Brdu；为胸腺嘧啶的衍生物，也是一种胸腺嘧啶核苷类似物，可代替胸腺嘧啶在DNA合成期(S期)，活体注射或细胞培养加入，而后利用抗Brdu单克隆抗体，ICC染色，显示增殖细胞。通常用作为细胞标记物，而最新研究表明其有新的功能。
作为本发明的优选方式，BrdU的分子结构式如以下式(VI)所示：
Figure PCTCN2016107910-appb-000009
如本发明所用，所述的EdU，中文名称：5-乙炔基-2’-脱氧尿苷；别名：5-乙炔基-2-脱氧尿苷；5-乙炔基-2’-去氧尿苷；乙炔基-脱氧尿苷；英语名称：5-Ethynyl-2’-deoxyuridine，简称为EdU；别名：EYdU；Uridine；5-Ethynyl-durd；5-Ethynyl-2’-dU；2’deoxyuridine；5-ethynyl；2’-deoxy-5-ethynyluridine；2’-deoxy-5-ethynyl-uridin；EdU是一种新型胸苷类似物，可掺入***中的细胞。较高剂量时会有细胞毒性。EdU可被一种荧光叠氮化物检出，后者通过点击化 学与前者形成共价键。不像常用的溴脱氧尿苷，EdU无需热或酸处理即可被检出。通常用作为细胞标记物，而最新研究表明其有新的功能。
作为本发明的优选方式，EdU的分子结构式如以下式(Ⅶ)所示：
Figure PCTCN2016107910-appb-000010
本发明还包括与上述化合物Ⅰ、Ⅱ或Ⅲ、Ⅳ、Ⅴ、Ⅵ或Ⅶ等效的化合物、药剂制品、类似物和/或其盐、水合物或前体；也包括其自然生成和人工合成化合物。
所述化合物的类似物包括但不限于：所述化合物的异构体、外消旋体。化合物具有一个或多个不对称中心。所以，这些化合物可以作为外消旋的混合物、单独的对映异构体、单独的非对映异构体、非对映异构体混合物、顺式或反式异构体存在。
所述的“盐”包括但不限于：(1)与如下无机酸形成的盐：如盐酸、硫酸、硝酸、磷酸等；(2)与如下有机酸形成的盐，如乙酸、草酸、丁二酸、酒石酸、甲磺酸、马来酸、或精氨酸等。其它的盐包括与碱金属或碱土金属(如钠、钾、钙或镁)形成的盐等。
所述的“化合物的前体”指当用适当的方法施用或处理后，该化合物的前体在培养基中，或动物，或人体内可转变成上述任一化合物的一种化合物，或上述任一化合物的一种化合物所组成的盐或溶液。
本发明组合物中，GSK3β抑制剂(或GSK3β抑制剂CHIR99021)、TGFβ抑制剂(或TGFβ抑制剂SB431542或/和A83-01)以重量份比：(0.046-4.65)﹕(0.038-7.68)；较佳地，(0.232-2.325)﹕(0.192-3.84)存在；或溶液状态下以摩尔比：(0.1-10)﹕(0.1-20)；较佳地，(0.5-5)﹕(0.5-10)存在。
该小分子组合物用于化学诱导人肿瘤细胞直接重编程转化为非致瘤性细胞并伴随肿瘤细胞凋亡；但该组合物偏重于诱导肿瘤细胞凋亡，转化肿瘤细胞功能偏弱；
作为本发明的优选方式，所述组合物还包括：维甲酸类化合物：0.03-6.0重量份；较佳地为：0.15-3.0重量份；或溶液状态下摩尔终浓度为：0.1-20uM；较佳地为0.5-10uM；添加上述成分，可促进和增强了肿瘤细胞的转化或凋亡，并扩大了适用肿瘤类型或范围。
所添加上述成分后的组合物中，GSK3β抑制剂(或GSK3β抑制剂CHIR99021)，TGFβ抑制剂(或TGFβ抑制剂SB431542或/和A83-01)，和维甲酸类化合物(或维甲酸)以重量份比：(0.046-4.65)﹕(0.038-7.68)﹕(0.03-6.0)；较佳地，(0.232-2.325)﹕(0.192-3.84)﹕(0.15-3)存在；或溶液状态下以摩尔比：(0.1-10)﹕(0.1-20)：(0.1-20)；较佳地，(0.5-5)﹕(0.5-10)﹕(0.5-10)存在。
在本发明所述的组合物中，还可添加包括选自下组的一种或两种成分：BMP抑制剂LDN-193189：0.02-4.65重量份；较佳地为：0.203-2.03重量份。或溶液状态下：终浓度为：0.05-10uM；较佳地为0.5-5uM；或/和BrdU，或/和EdU：0.15-30重量份(BrdU)，0.125-25重量份(EdU)；较佳地为：1.5-15重量份(BrdU)，1.25-12.5(EdU)；或溶液状态下：终浓度为：0.5-100uM(BrdU或EdU)；较佳地为5-50uM(BrdU或EdU)；添加上述成分，可进一步促进或增强诱导某些极度恶性肿瘤细胞的转分化或凋亡。
在另一优选例中，所述的组合物中，GSK3β抑制剂(如GSK3β抑制剂CHIR99021)，TGFβ抑制剂(如TGFβ抑制剂SB431542或/和A83-01)，维甲酸类化合物(如维甲酸)，BMP抑制剂(如BMP抑制剂LDN-193189)和BrdU以重量份比：(0.046-4.65)﹕(0.038-7.68)﹕(0.03-6.0)﹕(0.02-4.65)﹕0.15-30；较佳地，(0.232-2.325)﹕(0.192-3.84)﹕(0.15-3)﹕(0.203-2.03)﹕(1.5-15)存在；或溶液状态下以摩尔比：(0.1-10)﹕(0.1-20)﹕ (0.1-20)﹕(0.05-10)﹕(0.5-100)；较佳地，(0.5-5)﹕(0.5-10)﹕(0.5-10)﹕(0.5-5)﹕(5-50)存在。
在另一优选例中，所述的组合物中，GSK3β抑制剂(如GSK3β抑制剂CHIR99021)，TGFβ抑制剂(如TGFβ抑制剂SB431542或/和A83-01)，维甲酸类化合物(如维甲酸)，BMP抑制剂(如BMP抑制剂LDN-193189)和EdU以重量份比：(0.046-4.65)﹕(0.038-7.68)﹕(0.03-6.0)﹕(0.02-4.65)﹕(0.125-25)；较佳地，(0.232-2.325)﹕(0.192-3.84)﹕(0.15-3)﹕(0.203-2.03)﹕(1.25-12.5)存在；或溶液状态下以摩尔比：(0.1-10)﹕(0.1-20)﹕(0.1-20)﹕(0.05-10)﹕(0.5-100)；较佳地，(0.5-5)﹕(0.5-10)﹕(0.5-10)﹕(0.5-5)﹕(5-50)存在。
对于本发明所述的组合物的剂型没有特别的限制，可以是任何适用于哺乳动物服用的剂型；可制备的剂型包括：粉剂、散剂、片剂、丸剂、胶囊剂、缓释剂、控速释剂及其它固体剂型；注射剂、输液剂、混悬剂及其它液体剂型；以及气体剂型、半固体剂型等其它剂型。优选的，所述的剂型可以是但不限于：粉末剂、颗粒剂、胶囊、缓释剂、片剂等固体剂型或注射剂、输液剂、溶液剂、混悬液等液体剂型。
本发明的组合物的制备方法根据所需制备的剂型以及给药途径来决定，本领域技术人员在参考了本发明所提供的组合以及配比后，采用常规的药物组合物的制备方法即可制备出本发明的组合物。
应理解，尽管在具体实施方式中，本发明人列举了几种组合物形式，但本领域人员也可由此推导：本发明的其它任何一种组合形式也是同样具有突出效果的。
本发明人首次证实了本发明的小分子组合物可用于开发制备预防、改善或***的药物或药物配方。当用于预防、改善或***时，所用的组合物的有效剂量可随施用的模式和待***类型以及疾病的严重程度而变化。具体情况根据受试者的个体情况来决定，这在熟练医师或药剂师可以判断的范围内。
本发明中，所述的肿瘤或肿瘤细胞包括但不限于：肝癌、鼻咽癌、肺癌、胃癌、结直肠癌、胰腺癌、乳腺癌、卵巢癌、***癌、骨肉瘤、淋巴瘤、白血病、食管癌、***、口腔癌、唾液腺肿瘤、鼻腔与鼻旁窦恶性肿瘤、喉癌、耳部肿瘤、眼部肿瘤、甲状腺肿瘤、纵隔肿瘤、胸壁、胸膜肿瘤、小肠肿瘤、胆道肿瘤、胰腺与壶腹周围肿瘤、肠系膜与腹膜后肿瘤、肾脏肿瘤、肾上腺肿瘤、***、睾丸肿瘤、***癌、子宫内膜癌、卵巢恶性肿瘤、恶性滋养细胞肿瘤、外阴癌与***癌、恶性淋巴瘤、多发性骨髓瘤、软组织肿瘤、骨肿瘤、皮肤及附件肿瘤、恶性黑色素瘤或神经***肿瘤，以及其它血液***肿瘤和实质性肿瘤或其细胞。较佳地为肝癌或肝癌细胞。
培养基和试剂
本发明还提供了用于小分子组合物诱导人肿瘤细胞直接重编程(转分化)为非致瘤性细胞并伴随肿瘤细胞凋亡的培养基(以下简称：肿瘤细胞转分化伴随凋亡培养基)。
按本发明所提供的组合物终浓度配方，选择具体终浓度的小分子组合物进行配制。作为本发明的优选方式，将该具体小分子组合物中的不同成分，分别根据其溶质的不同性质和不同溶解度将其溶解于DMSO(二甲基亚砜)或其它有机溶剂或水性溶剂中配成浓缩液试剂(从1∶50-1∶10,000范围)；然后按该具体小分子组合物终浓度要求，将各小分子有机溶液浓缩液试剂稀释，添加入含10％小牛血清、1％青链霉素混合液(100x)的基础细胞培养基(或含有各种细胞因子或生长因子的无血清培养基)中，即可获得所述的肿瘤细胞转分化伴随凋亡培养基。其中，该培养基各组分的百分含量还可上下浮动50％；较佳地上下浮动30％；更佳地上下浮动20％，如10％，5％；(百分数均以v/v计)。
作为本发明的优选方式，所述的基础细胞培养基包括但不限于：DMEM/F12、MEM、DMEM、F12、IMDM、RPMI1640、Neuronal basal或Fischers等，均为市场上可购得的商品。
作为本发明的优选方式，所述“无血清培养基”指：不含血清而含有支持细胞增殖和生物反应的多种营养成分(如生长因子、组织提取物等)的细胞培养基。即将除血清以外的各种细胞因子或生长因子等添加剂，添加到基础细胞培养基中组成的细胞培养基。
作为本发明的优选方式，所述的含有各种细胞因子或生长因子的无血清培养基包括但不限于：ITS、N2、B27等，均为可自行配制或商购产品。
应理解，本领域技术人员熟悉所述的基础细胞培养基或无血清培养基的配制或购买途径，因此，基础细胞培养基或无血清培养基并不限于本发明中所举例的这些。
作为本发明的优选方式，所述的“肿瘤细胞转分化伴随凋亡培养基”具体配制如下实施：
(1)将①GSK3β抑制剂(或GSK3β抑制剂CHIR-99021)：0.046-4.65重量份；较佳地为：0.232-2.325重量份；或溶液状态下终浓度为0.1-10uM；优选量为：0.5-5uM；和②TGFβ抑制剂(或TGFβ抑制剂SB431542或/和A83-01)：0.038-7.68重量份；较佳地为：0.192-3.84重量份；或溶液状态下终浓度为0.1-20uM；优选量为：0.5-10uM混合，获得本发明用于化学诱导直接重编程人肿瘤细胞转化为非致瘤性细胞并伴随肿瘤细胞凋亡的小分子组合物。
(2)在(1)的组合物的基础上，还可添加包括：维甲酸类化合物(或维甲酸)：0.03-6.0重量份；较佳地为：0.15-3重量份；或溶液状态下终浓度为：0.1-20uM；较佳地为0.5-10uM；添加上述成分，可促进和增强了肿瘤细胞的转化或凋亡，并扩大了适用肿瘤类型或范围。
(3)在(2)的组合物基础上，还可添加包括选自下组的一种或两种成分：BMP抑制剂LDN-193189：0.02-4.65重量份；较佳地为：0.203-2.03重量份。
或溶液状态下：终浓度为：0.05-10uM；较佳地为0.5-5uM；或/和BrdU，或/和EdU： 0.15-30重量份(BrdU)，0.125-25(EdU)；较佳地为：1.5-15重量份(BrdU)，1.25-12.5(EdU)；或溶液状态下(BrdU或/和EdU)：终浓度为：0.5-100uM；较佳地为5-50uM。添加上述成分，可进一步促进或增强某些肿瘤细胞的转化或凋亡。
(4)将上述小分子组合物混合，可配制获得“肿瘤细胞转分化伴随凋亡培养基”。
本发明还提供了用于化学诱导人肿瘤细胞直接重编程(转分化)为非致瘤性细胞并伴随肿瘤细胞凋亡的实验动物注射或口服用试剂。
作为本发明的优选方式，将前述任一组合物中的各小分子组合物，按公斤体重计算出相应的用药量，将其溶于Captisol(1-30％)或Tween-80(5％)溶液中获得实验动物注射或口服用试剂；或取该组合物中不同组份的相应量的浓缩试剂，将其添加入注射用生理盐水或磷酸盐溶液(含有或不含5％FBS)中获得实验动物注射用或口服试剂。较佳地为溶于Captisol(1-30％)。
培养方法
本发明还公开了一种小分子组合物诱导人肿瘤细胞直接重编程(转分化)为非致瘤性细胞并伴随肿瘤细胞凋亡的方法，所述方法步骤包括：
(1)浓缩液试剂配制：根据权利要求1-6任一所述的组合物，将各成分溶解于有机溶剂或水性溶剂中配制成浓缩液试剂；较佳地，所述的有机溶剂包括二甲基亚砜；较佳地，所述的水性溶剂包括：水，生理盐水，磷酸盐缓冲液；
(2)培养基获得：将步骤(1)中的浓缩液试剂分别稀释入含5-20％小牛血清、1％青链霉素混合液(100x)的基础细胞培养基或含有各种细胞因子或生长因子的无血清培养基中(使得各组分的浓度符合权利要求1-6任一所述的组合物中所限定的浓度)，获得诱导肿瘤细胞转分化伴随凋亡的培养基；
其中，该培养基各组分的百分含量还可上下浮动50％；较佳地上下浮动30％；更佳地上下浮动20％，如10％，5％。
(3)诱导肿瘤细胞转分化并伴随凋亡：将肿瘤细胞在上述步骤(2)配制的“肿瘤细胞转分化伴随凋亡培养基”中混悬，铺板作为处理组；
将与处理组培养基溶解小分子组合物的溶剂(如DMSO或其它溶剂)等量地添加入同处理组所用含10％小牛血清、1％青链霉素混合液(100x)的基础细胞培养基(或含有各种细胞因子或生长因子的无血清培养基)中，获得“对照培养基”(百分数均以v/v计)；然后将与处理组等数量的肿瘤细胞加入“对照培养基”中混悬，铺板，作为对照组；
37℃培养，每2-4天换液一次；3-7天传代一次。
(4)诱导肿瘤细胞转分化并伴随凋亡的传代培养：弃原培养液，PBS洗涤一次，加入细胞消化液消化细胞，37℃，1-5分钟，终止细胞消化，离心，弃上清，将细胞沉淀重悬，按1:1-1:3传代铺板。按实验步骤第1和2项方法培养，每2-4天换液一次。所用消化液包括胰酶，EDTA,Acutase,TrypleE等。3-7天传代一次。
(5)诱导肿瘤细胞转分化并伴随凋亡，转分化获得正常(非致瘤性)细胞：经上述实验步骤(3)、(4)转分化伴随凋亡培养和传代培养肿瘤细胞1-3周，PBS洗涤去除凋亡细胞，即可获得非致瘤性细胞。可用该细胞进行其他科研实验；细胞凋亡的检测：肿瘤细胞的转分化伴随凋亡培养如上述培养方法实验步骤。培养不同时间的肿瘤细胞用Annexin V-FITC detection kit(Biovision)染色后进行流式细胞检测，具体步骤见试剂盒说明书。
转分化获得的非致瘤性细胞的功能检测：肿瘤细胞的转分化伴随凋亡培养如上述，用培养不同时间获得的非致瘤性细胞检测其相关功能。
本发明的“肿瘤细胞转分化伴随凋亡小分子组合物”及其制备的培养基和试剂以及其实验方法，不仅可研发制备***药物，还可广泛用于防治肿瘤方法及机理研究、临床前期研究、药理及毒理安全性检测；所获得的非致瘤性细胞可继续进行功能检测、致瘤性多靶点实验、临床前期研究等。该方法不仅为防治肿瘤研究开辟了一个崭新领域，具有广泛的应用前景；而且丰富了干细胞重编程理论，拓展其应用范围。具有重大的科学意义和极大的应用价值。
本发明的有益效果在于：
1.率先借鉴细胞重编程机理，提出：筛选能够构建并维持重编程目的细胞的特有基因表达谱或生物学行为特性，以及能够突破起始细胞重编程过程中的各种“能障”的诱导因子组合，就可以将肿瘤细胞直接转分化为目的细胞，这一创新思路。
2.率先将细胞重编程机理及化学诱导细胞直接重编程(转分化)方法应用于肿瘤治疗研究并取得预期结果。使用小分子诱导肝癌细胞转分化为具功能(体外)的非致瘤性肝细胞并伴随癌细胞凋亡；相似思路和方法应用于其他肿瘤细胞转分化也获得相同或相似效果。
3.率先将化学诱导肝癌细胞转分化方法应用于体内肿瘤转分化(肝癌组织PDX动物模型实验)。结果显示：体内大部分肝癌组织坏死，未坏死的组织不再具有肝癌的组织结构，其中的细胞高表达HNF4a，提示其已转分化。证明本发明的小分子组合物，对病人的肝癌治疗有潜在疗效；
4.本发明中，应用的小分子性质稳定，作用的时间、剂量及组合方式易于控制，作用效果稳定可靠；
5.本发明无外源基因导入，不改变细胞的基因结构，避免外源基因导入或结构基因改变引起新的致癌风险，因此安全可靠；
6.率先提出使用多靶点诱导肿瘤细胞转分化为正常(非致瘤性)细胞，并且不使用杀细胞制剂辅助控制或者***。因此对正常细胞无损伤(如正常人成纤维细胞和肝细胞)，避免了对正常细胞的损伤及毒副作用；
7.率先使用小分子组合诱导肿瘤细胞直接重编程为非致瘤性细胞，而不经过重编程为诱导多能干细胞(iPSC)阶段，避免了干细胞体内致癌风险；
8.本发明的小分子组合物不仅对诱导肝癌细胞转分化伴随凋亡有效，进一步的研究结果显示，对诱导鼻咽癌、肺癌、胃癌、结直肠癌、胰腺癌、乳腺癌、卵巢癌、***癌、骨肉瘤、淋巴瘤、白血病及其他血液***肿瘤和实质性肿瘤细胞的转分化为非致瘤性细胞，并伴随凋亡均有相同或相似效果；
9.本发明的小分子组合物有潜力开发成高效低毒的控制或治疗肝癌以及其他恶性肿瘤的新方法、新手段、新药物；或制备成化学诱导肿瘤细胞直接重编程为非致瘤性细胞的科研用培养基和试剂；
10.本发明小分子组合物及其诱导肿瘤细胞转分化伴随凋亡方法操作简单，成本低廉，易于生产应用。
11.本发明丰富了重编程理论，拓宽其应用范围；开创了肿瘤治疗研究新领域；为临床肿瘤治疗提供新思想、新方法、新药物。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如J.萨姆布鲁克等编著，分子克隆实验指南，第三版，科学出版社，2002中所述的条件，或按照制造厂商所建议的条件。
实施例1、肿瘤细胞转分化伴随凋亡小分子组合物及其培养基和试剂的配制
如下配制组合物或培养基，可按摩尔浓度或重量浓度进行配制：
1、肿瘤细胞转分化伴随凋亡小分子组合物的配方设计
设计如下配方的组合物：
(1)肿瘤细胞转分化伴随凋亡组合物1
GSK3β抑制剂CHIR-99021：终浓度2uM；
TGFβ抑制剂SB431542：终浓度5uM。
(2)肿瘤细胞转分化伴随凋亡组合物2
GSK3β抑制剂CHIR-99021：终浓度3uM；
TGFβ抑制剂SB431542：终浓度2uM。
(3)肿瘤细胞转分化伴随凋亡组合物3
GSK3β抑制剂CHIR-99021：终浓度5uM；
TGFβ抑制剂A83-01：终浓度2uM。
(4)肿瘤细胞转分化伴随凋亡组合物4
GSK3β抑制剂CHIR-99021：终浓度4uM；
TGFβ抑制剂A83-01终浓度3uM；
维甲酸(RA)：终浓度0.5uM。
(5)肿瘤细胞转分化伴随凋亡组合物5
GSK3β抑制剂CHIR-99021：终浓度3uM；
TGFβ抑制剂SB431542：终浓度5uM；
维甲酸(RA)：终浓度3uM。
(6)肿瘤细胞转分化伴随凋亡组合物6
GSK3β抑制剂CHIR-99021：2uM；
TGFβ抑制剂SB431542：终浓度2uM；
维甲酸(RA)：终浓度5uM。
(7)肿瘤细胞转分化伴随凋亡组合物7
GSK3β抑制剂CHIR-99021：终浓度3uM；
TGFβ抑制剂SB431542：终浓度1uM；
维甲酸(RA)：终浓度0.5uM；
BMP抑制剂LDN-193189：终浓度0.5uM。
(8)肿瘤细胞转分化伴随凋亡组合物8
GSK3β抑制剂CHIR-99021：终浓度3uM；
TGFβ抑制剂SB431542：终浓度5uM；
维甲酸(RA)：终浓度3uM；
BMP抑制剂LDN-193189：0.5uM。
(9)肿瘤细胞转分化伴随凋亡组合物9
GSK3β抑制剂CHIR-99021：终浓度3uM；
TGFβ抑制剂SB431542：终浓度2uM；
维甲酸(RA)：终浓度10uM；
BMP抑制剂LDN-193189：终浓度0.5uM。
(10)肿瘤细胞转分化伴随凋亡组合物10
GSK3β抑制剂CHIR-99021：终浓度3uM；
TGFβ抑制剂SB431542：终浓度5uM；
BMP抑制剂LDN-193189：2uM。
(11)肿瘤细胞转分化伴随凋亡组合物11
GSK3β抑制剂CHIR-99021：终浓度3uM；
TGFβ抑制剂SB431542：终浓度7.5uM；
BMP抑制剂LDN-193189：终浓度0.5uM。
(12)肿瘤细胞转分化伴随凋亡组合物12
GSK3β抑制剂CHIR-99021：终浓度5uM；
TGFβ抑制剂SB431542：终浓度2uM；
维甲酸(RA)：终浓度5uM。
(13)肿瘤细胞转分化伴随凋亡组合物13
GSK3β抑制剂CHIR-99021：终浓度4uM；
TGFβ抑制剂A83-01终浓度3uM；
维甲酸(RA)：终浓度0.5uM；
BrdU：终浓度15uM。
(14)肿瘤细胞转分化伴随凋亡组合物14
GSK3β抑制剂CHIR-99021：终浓度3uM；
TGFβ抑制剂SB431542：终浓度5uM；
维甲酸(RA)：终浓度3uM；
BMP抑制剂LDN-193189：0.5uM；
EdU：终浓度30uM。
(15)肿瘤细胞转分化伴随凋亡组合物15
GSK3β抑制剂BIO：终浓度3uM；
TGFβ抑制剂SB431542：终浓度7.5uM；
BMP抑制剂LDN-193189：终浓度0.5uM。
(16)肿瘤细胞转分化伴随凋亡组合物16
GSK3β抑制剂CHIR-99021：终浓度5uM；
TGFβ抑制剂LY2109761：终浓度2uM；
维甲酸(RA)：终浓度5uM。
(17)肿瘤细胞转分化伴随凋亡组合物17
GSK3β抑制剂CHIR-99021：终浓度3uM；
TGFβ抑制剂RepSox：终浓度7.5uM；
BMP抑制剂LDN-193189：终浓度0.5uM。
(18)肿瘤细胞转分化伴随凋亡组合物18
GSK3β抑制剂CHIR-98014：终浓度3uM；
TGFβ抑制剂A83-01：终浓度5uM；
BMP抑制剂LDN-193189：2uM。
(19)肿瘤细胞转分化伴随凋亡组合物19
GSK3β抑制剂CHIR-99021：终浓度4uM；
TGFβ抑制剂SB431542：终浓度3uM；
9-顺式维甲酸：终浓度0.5uM。
(20)肿瘤细胞转分化伴随凋亡组合物20
GSK3β抑制剂TWS119：终浓度4uM；
TGFβ抑制剂A83-01：终浓度3uM；
维甲酸(RA)：终浓度0.5uM。
各具体小分子组合物按前述“培养方法”步骤(1)先溶解于DMSO中制成浓缩液试剂。
2、肿瘤细胞转分化伴随凋亡培养基配制
将上述实验步骤1配制的肿瘤细胞转分化伴随凋亡组合物1～20各成分DMSO浓缩液试剂按前述培养方法步骤(2)配制(选用的基础培养基是DMEM/F12)，获得肿瘤细胞转分化伴随凋亡培养基1～20(即培养基1与组合物1的化合物终浓度相同，培养基2与组合物2的化合物终浓度相同，…，培养基20与组合物20的化合物终浓度相同)。
3、肿瘤细胞转分化伴随凋亡注射或口服用试剂配制
将上述实验步骤1配制的“肿瘤细胞转分化伴随凋亡组合物8”的各成分溶于1％Captisol,获得实验动物注射或口服用试剂8。
按mg/kg(体重)计算配制“肿瘤细胞转分化伴随凋亡组合物21”：
GSK3抑制剂CHIR-99021：1mg/kg；
TGFβ抑制剂SB431542：0.5mg/kg；
维甲酸(RA)：0.2mg/kg；
BMP抑制剂LDN-193189：0.2mg/kg；
将上述各成分溶于1％Captisol,获得实验动物注射或口服用试剂21。
实施例2、肝癌细胞SMMC-7721被培养基6诱导转分化伴随凋亡
1、肿瘤细胞转分化伴随凋亡培养
将肝癌细胞SMMC-7721在上述已经配制的培养基6中混悬，铺板作为处理组。
将与处理组等量的DMSO(二甲基亚砜)添加入含10％小牛血清、1％青链霉素混合液(100x)的基础细胞培养基DMEM/F12中，获得DMSO“对照培养基”(百分数均以v/v计)；然后将与处理组等数量的肝癌细胞SMMC-7721加入DMSO“对照培养基”中混悬，铺板，作为对照组；
37℃培养，每2-4天换液一次；3-7天传代一次。
2、转分化伴随凋亡培养的肝癌细胞的传代培养
传代培养步骤：弃原培养液，PBS洗涤一次，加入细胞消化液消化细胞，37℃，1-5分钟，终止细胞消化，离心，弃上清，将细胞沉淀重悬，按1:1-1:3传代铺板。按实验步骤第1和2项方法培养，每2-4天换液一次。所用消化液是胰酶(也可用EDTA，Acutase，TrypleE)等。3-7天传一次代。
3、转分化伴随凋亡培养获得肝细胞样细胞
经实验步骤1、2的转分化伴随凋亡培养以及传代培养肝癌细胞1-3周，离心去除凋亡细胞后，未凋亡的肝癌细胞被转分化为非致瘤性肝细胞样细胞；获得的非致瘤性肝细 胞样细胞，可进行其他科研实验。
实验结果见图1。A图、肝癌细胞SMMC-7721被诱导转分化，形态完全改变，显示已被转分化；B图、肝癌细胞SMMC-7721被诱导转分化伴随凋亡的统计结果。处理组在不同处理时间段都存在不同程度的早期和晚期凋亡，对照组几乎无早期凋亡，有部分晚期自然凋亡。
实施例3、小分子组合(培养基1、4)诱导肝癌细胞HepG2转分化伴随凋亡
处理组和对照组设置以及培养实验步骤同实施例2，不同点在于以培养基1、4分别取代培养基6。
培养处理1-3周的实验结果见图2。A图、肝癌细胞HepG2被(培养基4)诱导转分化，形态完全改变，显示已被转分化；除早、晚期被诱导凋亡的肝癌细胞以外，剩下的肝癌细胞全部转分化。B图、肝癌细胞HepG2被(培养基1)诱导凋亡统计；处理组在不同处理时间段都存在不同程度的早期和晚期凋亡，对照组几乎无早期凋亡，有一定的晚期自然凋亡。
实施例4、耐5-Fu肝癌细胞7402/5-Fu分别被培养基5、2诱导转分化并伴随凋亡
处理组和对照组设置以及培养实验步骤同实施例2，不同点在于以肿瘤细胞转分化伴随凋亡培养基5或肿瘤细胞转分化伴随凋亡培养基2取代肿瘤细胞转分化伴随凋亡培养基6。
培养处理1-4周的实验结果见图3。A、右图肝癌细胞7402/5-Fu被(培养基5)诱导转分化为肝细胞样细胞，形态完全改变，显示已被转分化；B、肝癌细胞7402/5-Fu被(培养基2)诱导转分化伴随凋亡统计。处理组在不同处理时间段都存在不同程度的早期和晚期凋亡，对照组无早期凋亡，有极少晚期自然凋亡。
实施例5、肝癌细胞SMMC-7721，HepG2，7402/5-Fu分别被培养基6、4、5诱导转分化为具有正常肝细胞功能的肝细胞样细胞
处理组和对照组设置以及培养实验步骤同实施例2、3和4，采用肿瘤细胞转分化伴随凋亡培养基6、4、5。培养处理2周的处理组离心去除凋亡细胞后，未凋亡的肝癌细胞被转分化为非致瘤性肝细胞样细胞。对照组培养同样时间收取细胞。
(1)肝糖原染色
用Schiff方法。具体实验步骤为：①弃细胞培养液，PBS漂洗1次，②4％多聚甲醛固定10分钟后，PBS漂洗5分钟×3次，③加入PAS-I液10min，流水冲洗，④加入PAS-II液1-2min，流水冲洗，⑤显微镜观察并拍照。
(2)油红染色(Oil-red)
用试剂盒检测。具体实验步骤见试剂盒说明书。试剂盒购自南京建成科技有限公 司，货号：D027。
实验结果见图4。肝癌细胞SMMC-7721(培养基6)，HepG2(培养基4)，7402/5-Fu(培养基5)分别被诱导转分化后，所获得的肝细胞样细胞具有正常肝细胞功能。PAS：糖原染色，Oil-red：油红染色，反映脂肪摄取功能。
因此，如上肝癌细胞转分化后获得的肝细胞样细胞具有正常人肝细胞相关功能。
实施例6、肝癌细胞SMMC-7721、HepG2、7402/5-Fu分别被培养基10、11、12诱导转分化为具肝细胞功能的细胞
处理组和对照组设置以及培养实验步骤同实施例2，采用肿瘤细胞转分化伴随凋亡培养基10、11、12。培养处理2周的处理组离心去除凋亡细胞后，收取处理组和对照组的细胞培养上清液及细胞。
(1)白蛋白分泌(ALB)，尿素生成(Urea)测试
用ELISA方法，用试剂盒分别检测处理组和对照组培养的细胞的白蛋白分泌和尿素生成功能。具体实验步骤见试剂盒说明书。白蛋白检测试剂盒(美国Bioassay System公司/DIAG-250，BCG Albumin assay kit)，尿素检测试剂盒(美国Bioassay System公司/DIUR-500，Urea assay kit)。
(2)P450酶(CYP3A4和CYP1A2)活性诱导测试
用利福平(诱导CYP3A4)和奥美拉措(诱导CYP1A2)分别诱导不同的P450酶活性。不同浓度的利福平(1uM、10uM、25uM)或奥美拉措(1uM、10uM、25uM)分别加入处理组和对照组的细胞培养液中培养48小时，然后收取细胞RNA，用qRT-PCR定量检测不同处理条件的细胞的CYP3A4和CYP1A2基因表达量。
实验结果见图5。
A、肝癌细胞SMMC-7721被(培养基10)诱导转分化，所获得的肝细胞样细胞具有正常肝细胞功能；
B、肝癌细胞HepG2被(培养基11)诱导转分化，所获得的肝细胞样细胞具有正常肝细胞功能；
C、耐5-Fu肝癌细胞7402/5-Fu被(培养基12)诱导转分化，所获得的肝细胞样细胞具有正常肝细胞功能；
图中蓝色柱代表早期凋亡，红色柱代表晚期凋亡；T1W、T2W、T3W分别代表处理1、2、3周；Rif：利福平；Ome：奥美拉措。
因此，转分化获得的肝细胞样细胞具有正常人肝细胞相关功能，被转分化的肝癌细胞获得正常肝细胞白蛋白分泌(ALB)，尿素生成(Urea)，CYP1A2诱导和CYP3A4诱导功能。
实施例7、肝癌细胞SMMC-7721，HepG2和7402/5-Fu分别被(培养基6、4、5) 诱导转分化，所获得的肝细胞样细胞体内体外均不再具致瘤性。
处理组和对照组设置以及培养实验步骤同实施例2，采用肿瘤细胞转分化伴随凋亡培养基6、4、5分别培养处理2周，处理组去除凋亡细胞后，未凋亡的肝癌细胞被转分化为肝细胞样细胞；获得的肝细胞样细胞体内体外均无致瘤性。
1、转分化获得的肝细胞样细胞体外致瘤性实验
方法步骤：分别将处理组和对照组1×103个细胞，种于6孔板，培养方式同实施例2，培养2周；结晶紫染色，拍照片，观察并计数细胞生长形成克隆。结果见图6。图A：肝癌细胞SMMC-7721(培养基6处理)，HepG2(培养基4处理)和7402/5-Fu(培养基5处理)被转分化后获得的肝细胞样细胞体外生长不形成克隆，体外无致瘤性。
2、转分化获得的肝细胞样细胞体内皮下荷瘤实验
取处理20天肝癌细胞转分化获得的肝细胞样细胞，用消化液消化为单个细胞，离心，PBS漂洗，细胞计数，取1×106的细胞，注射到5周龄大小的裸鼠后腿皮下(右侧)，用DMSO“对照培养基”处理相同时间的癌细胞作为对照注射到同一只裸鼠的后腿皮下(左侧)，4周终止实验，分离瘤体并拍照。结果见图6。图B：肝癌细胞SMMC-7721被培养基6诱导转分化，获得的肝细胞样细胞体内不生成肿瘤，失去致瘤性。图C：7402/5-Fu被培养基8诱导转分化，获得的肝细胞样细胞体内不生成肿瘤，失去致瘤性；C图上图裸鼠右侧处理组不形成肿瘤；左侧对照组形成肿瘤；C图下图为对照组形成的肿瘤解剖形状外观。
实施例8、病人肝癌组织PDX动物模型试验(转分化伴随凋亡小分子组合物8)
取肝癌病人手术切除的瘤组织植入5周龄大小的裸鼠皮下成瘤(PDX模型)。分离裸鼠皮下成瘤的瘤体，再次植入5周龄大小的裸鼠皮下成瘤。待肿瘤生长到5-10mm大小时进行药物处理。将实施例1配制的肿瘤细胞转分化伴随凋亡实验动物注射用试剂8进行瘤体注射，3次/周，用DMSO生理盐水注射液瘤体注射为对照。4周后终止实验。分离瘤体，拍照，固定瘤组织，HE染色。
实验结果见图7。处理组瘤体组织及细胞坏死，组织结构已被破坏或丧失；对照组瘤体组织、细胞结构无变化。
实施例9、正常人成纤维细胞和肝细胞分别被肿瘤细胞转分化伴随凋亡培养基8、3处理培养的情况
处理组和对照组设置及实验培养方法同实施例2，不同点在于依次以肿瘤细胞转分化伴随凋亡培养基8或3分别培养正常人成纤维细胞和肝细胞。
结果见图8，正常人成纤维细胞和肝细胞被肿瘤细胞培养3周后，和对照组相比形态无改变，显示其不受影响。
实施例10、鼻咽癌细胞HNE和肺癌细胞H460被培养基9、7诱导转分化并伴随凋亡
处理组和对照组设置以及培养实验步骤同实施例2。培养处理2周的实验结果见图9。
鼻咽癌细胞HNE(培养基9处理)处理组癌细胞转分化后，形态完全改变，显示其已转分化；处理组肺癌细胞H460被(培养基9、7)分别处理，其中肺癌细胞H460被(培养基7)几乎全部诱导凋亡；而对照组肺癌细胞H460几乎无凋亡。
实施例11、胃癌细胞SGC-7901和MKN28分别被(培养基12、13)诱导转分化并伴随凋亡
处理组和对照组设置以及培养实验步骤同实施例2。
培养处理2周的实验结果见图10，处理组的胃癌细胞SGC-7901(培养基12)和MKN28,(培养基13)转分化后，细胞形态完全改变，显示其已被转分化。
实施例12、胰腺癌细胞SW1990被培养基12诱导转分化
处理组和对照组设置以及培养实验步骤同实施例2。
胰腺癌细胞SW1990被(培养基12)处理2周的实验结果见图11，处理组的癌细胞转分化后，形态完全改变，显示其已转分化。
实施例13、乳腺癌细胞SKBr3被培养基13诱导转分化
处理组和对照组设置以及培养实验步骤同实施例2。培养处理2周(培养基13)的实验结果见图12，处理组的乳腺癌细胞SKBr3转分化后，形态完全改变，显示其已转分化。
实施例14、白血病细胞U937、B细胞淋巴瘤SUDHL-4被培养基10、11诱导转分化并伴随凋亡
处理组和对照组设置以及培养实验步骤同实施例2。培养处理2周的实验结果见图13，处理组的白血病细胞U937(培养基10培养)、B细胞淋巴瘤细胞SUDHL-4(培养基11培养)大量凋亡。
实施例15、乳腺癌细胞SKBr3和胃癌细胞MKN28被培养基13、14诱导转分化
处理组和对照组设置以及培养实验步骤同实施例2。培养处理2周的实验结果见图14，处理组乳腺癌细胞SKBr3(培养基13)和胃癌细胞MKN28(培养基14)转分化后，细胞形态完全改变，显示其已转分化。
实施例16、肠癌细胞HCT116被培养基9诱导转分化为非致瘤性细胞
处理组和对照组设置以及培养实验步骤同实施例2。培养处理2周的实验结果见图15，右图显示处理组的肠癌细胞HCT116(培养基9)转分化后，细胞不再形成克隆，失去致瘤性。
实施例17、***癌细胞PC-3，卵巢癌细胞SKOV3和A2780分别被培养基5、7、8诱导转分化后失去致瘤性
处理组和对照组设置以及培养实验步骤同实施例7。处理2周后，实验结果见图16、17。图16右图、图17右上下图分别显示处理组的***癌PC-3(培养基5)，卵巢癌细胞SKOV3(培养基7)和A2780(培养基8)被诱导转分化后不再形成克隆，失去体外致瘤性。
实施例18、胃癌细胞MKN28，乳腺癌细胞SKbr3分别被培养基10、11诱导转分化后失去致瘤性。
处理组和对照组设置以及培养实验步骤同实施例7，处理2周后，实验结果见图18。图18右上下图分别显示处理组的胃癌细胞MKN28(培养基10处理)和乳腺癌细胞SKbr3(培养基11处理)转分化后不再形成克隆，失去体外致瘤性。
实施例19、神经胶质瘤细胞T98G、U87MG被培养基4、5诱导转分化并伴随凋亡
处理组和对照组设置以及培养实验步骤同实施例14，培养处理2周的实验结果见图19。图19右上下图处理组神经胶质瘤细胞T98G(培养基4处理)、U87MG(培养基5处理)被分别诱导转分化后形态完全改变，显示其已转分化。
实施例20、病人肝癌组织PDX动物模型试验(肿瘤细胞转分化伴随凋亡小分子组合物15)
实验动物注射试剂，处理组和对照组设置以及实验步骤，染色方法同实施例8，不同之处是使用肿瘤细胞转分化伴随凋亡组合物15配制的注射用试剂15，实验结果见图20。
图20左图处理组的肝癌组织及细胞，经注射用试剂15处理3周后，大面积坏死，癌组织结构已被破坏或丧失。右图处理组显示，经小分子试剂处理3周后，残留组织及细胞表达人肝细胞特有标志物HNF4a，提示已发生转分化。
实施例21、肺癌细胞A549、H1299和H460分别被培养基14、13、9诱导转分化后，体内外不再具致瘤性
实验动物注射试剂，处理组和对照组设置以及动物实验步骤等，均同实施例7，实验结果见图21。
A图：培养处理2周结果，下图处理组显示，肺癌细胞A549(培养基14)、H1299(培养基13处理)和H460(培养基9处理)分别被诱导转分化后，不再形成克隆，体外失去致瘤性；B图：裸鼠右侧后腿(蓝色箭头所指)为处理组肺癌细胞A549(培养基14处理)转分化后注射入裸鼠体内4周，不再形成肿瘤，体内失去致瘤性。
实施例22、肺癌细胞H1299被培养基15诱导转分化为非致瘤性细胞
处理组和对照组设置以及培养实验步骤同实施例2。
培养处理2周的实验结果见图22，右图显示处理组的肺癌细胞H1299细胞(培养基15处理)转分化后不再形成克隆，失去致瘤性。
表明GSK3β抑制剂的其他小分子BIO与TGFβ抑制剂及BMP抑制剂构成的小分子组合，也有同样的诱导肿瘤细胞转分化伴随凋亡的效果。
实施例23、卵巢癌细胞A2780、SKOV3被(培养基16、17)分别诱导转分化为非致瘤性细胞
处理组和对照组设置以及培养实验步骤同实施例2。
培养处理2周的实验结果见图23，右上下图分别显示处理组的卵巢癌细胞A2780(培养基16处理)、SKOV3(培养基17处理)转分化后不再形成克隆，失去致瘤性。
表明TGFβ抑制剂的其他小分子LY2109761或RepSox与GSK3β抑制剂和维甲酸类化合物或BMP抑制剂组成的小分子组合物，也有同样的诱导肿瘤细胞转分化伴随凋亡效果。
实施例24、***癌细胞PC9被(培养基18)诱导转分化
处理组和对照组设置以及培养实验步骤同实施例2
培养处理(培养基18)2周的实验结果见图24，右图处理组的***癌细胞PC9，细胞形态完全改变，显示其已转分化。
表明GSK3β抑制剂的其他小分子CHIR-98014与TGFβ抑制剂及BMP抑制剂组成的小分子组合物，也有同样的诱导肿瘤细胞转分化伴随凋亡效果。
实施例25、胃癌SGC-7901细胞被培养基19、20分别诱导转分化并伴随凋亡
1、胃癌SGC-7901细胞被培养基19诱导转分化后失去致瘤性
处理组和对照组设置以及培养实验步骤同实施例2。处理2周后，实验结果见图25(A图)。A图处理组显示胃癌SGC-7901细胞，转分化后不再形成克隆，体外失去致 瘤性。
表明维甲酸类化合物的其他小分子9-顺式维甲酸与GSK3β抑制剂、TGFβ抑制剂组成的小分子组合物，也有同样的诱导肿瘤细胞转分化伴随凋亡的效果。
2、胃癌SGC-7901细胞被培养基19、20诱导转分化伴随凋亡
处理组和对照组设置以及培养实验步骤同实施例2。
培养处理2周，胃癌SGC-7901细胞被诱导转分化伴随凋亡的统计结果见图25。B图：处理组在不同处理时间段都存在不同程度的早期和晚期凋亡，对照组有很少部分早、晚期自然凋亡。对照组和处理组的凋亡值具统计学差异(p<0.005)。
表明GSK3β抑制剂的其他小分子TWS119与TGFβ抑制剂及维甲酸类化合物组成的小分子组合物，也有同样的诱导肿瘤细胞转分化伴随凋亡效果。
实施例26、胰腺癌SW1990细胞被培养基16诱导转分化后失去致瘤性
处理组和对照组设置以及培养实验步骤同实施例2。处理2周后，实验结果见图26。右图处理组的胰腺癌SW1990细胞(培养基16处理)被诱导转分化后不再形成克隆，体外失去致瘤性。
表明TGFβ抑制剂的其他小分子LY2109761与GSK3β抑制剂和维甲酸类化合物组成的小分子组合物，也有同样的诱导肿瘤细胞转分化伴随凋亡效果。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (12)

  1. 一种用于化学诱导人肿瘤细胞直接重编程转化或转分化为非致瘤性细胞并伴随肿瘤细胞不同程度凋亡的小分子组合物，其特征在于，所述的组合物包括GSK3β抑制剂，TGFβ抑制剂；或所述的组合物由GSK3β抑制剂，TGFβ抑制剂组成。
  2. 如权利要求1所述的组合物，其特征在于，所述的组合物包括：
    GSK3β抑制剂：0.046-4.65重量份；或溶液状态下终浓度为：0.1-10uM；和
    TGFβ抑制剂：0.038-7.68重量份；或溶液状态下终浓度为：0.1-20uM。
  3. 如权利要求2所述的组合物，其特征在于，所述的组合物包括：
    GSK3β抑制剂：0.232-2.325重量份；或溶液状态下终浓度为：0.5-5uM；和
    TGFβ抑制剂：0.192-3.84重量份；或溶液状态下终浓度为：0.5-10uM。
  4. 如权利要求1所述的组合物，其特征在于，所述组合物还包括：
    维甲酸类化合物：0.03-6.0重量份；较佳地为0.15-3重量份；或溶液状态下终浓度为：0.1-20uM；较佳地为0.5-10uM。
  5. 如权利要求1所述的组合物，其特征在于，所述组合物还可添加包括选自下组的一种或多种成分：
    BMP抑制剂：0.02-4.65重量份；较佳地为：0.203-2.03重量份；或溶液状态下终浓度为：0.05-10uM；较佳地为0.5-5uM；或
    BrdU：0.15-30重量份；较佳地1.5-15重量份；或溶液状态下终浓度为：0.5-100uM；较佳地为5-50uM；或
    EdU：0.125-25重量份；较佳地1.25-12.5重量份；或溶液状态下终浓度为：0.5-100uM；较佳地为5-50uM。
  6. 如权利要求1-5任一所述的组合物，其特征在于，所述的GSK3β抑制剂包括：CHIR-99021、BIO、IM-12、TWS119、1-Azakenpaullone、CHIR-98014、Tideglusib、AR-A014418、LY2090314、SB216763、AZD1080等为代表的具相同功能，或诱导相同靶点的同一类GSK3β信号通路抑制剂或化合物，或与它们等效的药剂制品、类似物、异构体和/或其盐、水合物或前体，或其组合；较佳地为GSK3β抑制剂CHIR-99021；
    所述的TGFβ抑制剂包括：SB431542、A83-01、SB525334、LY2109761，RepSox、SD-208、GW788388、SB505124、EW-7197等为代表的，具相同功能，或诱导相同靶点的同一类TGFβ信号通路抑制剂或化合物，或与它们等效的药剂制品、类似物、异构体和/或其盐、水合物或前体，或其组合；较佳地为TGFβ抑制剂SB431542或/和A83-01；
    所述的维甲酸类化合物是天然或人工合成的，包括：维甲酸、13-顺式维甲酸、9-顺式维甲酸、异维甲酸等为代表的，具相同功能，或诱导相同靶点的同一类维甲酸类诱导分化剂或化合物，或与它们等效的药剂制品、类似物、异构体和/或其盐、水合物或前体，或其组合；较佳地为维甲酸；
    所述的BMP抑制剂包括：LDN-193189、K02288、DMH1等为代表的，具相同功能，或诱导相同靶点的同一类BMP信号通路抑制剂或化合物，或与它们等效的药剂制品、类似物、异构体和/或其盐、水合物或前体，或其组合；较佳地为BMP抑制剂LDN-193189。
  7. 如权利要求1-5任一所述的组合物，其特征在于，所述的组合物是药物组合物，还包含药学上可接受的载体或赋形剂，其载体或赋形剂包括：
    水、盐水、磷酸缓冲液或其它水性溶剂；
    DMSO、甘油和乙醇或其它有机溶剂；
    微球、脂质体、微乳液或高分子表面活性剂；
    胶体型载药***或高分子载药***；或
    防腐剂、抗氧剂、矫味剂、芳香剂、助溶剂、乳化剂、pH缓冲物质，黏合剂、填充剂、润滑剂或其它药物赋形剂。
  8. 如权利要求1-5任一所述的组合物，其特征在于，所述的组合物能制备的药物剂型包括：
    固体剂型，包括粉剂、散剂、片剂、丸剂、胶囊剂、缓释剂、控速释剂，或其他固体剂型；
    液体剂型，包括注射剂、输液剂、混悬剂，或其它液体剂型；
    以及气体剂型；或
    半固体剂型。
  9. 权利要求1-8任一所述的组合物的用途，其特征在于，用于研发或制备***的药物；或用于制备诱导人肿瘤细胞直接重编程转化或转分化为非致瘤性细胞并伴随肿瘤细胞凋亡的培养基或试剂。
  10. 一种诱导肿瘤细胞直接重编程转化或转分化为非致瘤性细胞并伴随肿瘤细胞凋亡的方法，其特征在于，所述方法包括：应用权利要求1-8任一所述的组合物处理人肿瘤细胞，使之直接重编程为非致瘤性细胞，并伴随肿瘤细胞不同程度凋亡。
  11. 一种用于诱导人肿瘤细胞直接重编程转化或转分化为非致瘤性细胞并伴随肿瘤细胞凋亡的药盒/试剂盒，其特征在于，所述的药盒/试剂盒中包括：权利要求1-8任一所述的组合物；或基于该组合物开发制备的***的药物或药物配方；或基于该组合物制备的试剂或培养基。
  12. 如权利要求1-8任一所述的组合物、权利要求9所述的用途、权利要求10所述的方法、权利要求11所述的药盒或试剂盒，其特征在于，所述的肿瘤或肿瘤细胞包括但不限于：肝癌、肺癌、胃癌、结直肠癌、胰腺癌、乳腺癌、卵巢癌、***癌、骨肉瘤、淋巴瘤、白血病、鼻咽癌、食管癌、***、口腔癌、唾液腺肿瘤、鼻腔与鼻旁窦恶性肿瘤、喉癌、耳部肿瘤、眼部肿瘤、甲状腺肿瘤、纵隔肿瘤、胸壁、胸膜肿瘤、小肠肿瘤、胆道肿瘤、胰腺与壶腹周围肿瘤、肠系膜与腹膜后肿瘤、肾脏肿瘤、 肾上腺肿瘤、***、睾丸肿瘤、***癌、子宫内膜癌、卵巢恶性肿瘤、恶性滋养细胞肿瘤、外阴癌与***癌、恶性淋巴瘤、多发性骨髓瘤、软组织肿瘤、骨肿瘤、皮肤及附件肿瘤、恶性黑色素瘤或神经***肿瘤及其它血液***肿瘤和实质性肿瘤或其细胞；较佳地为肝癌或肝癌细胞。
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